Antibodies capable of binding to cd27, variants thereof and uses thereof

ABSTRACT

The present invention relates to antibodies capable of binding to human CD27 and to variants thereof comprising a modified Fc region comprising one or more mutations that enhances the Fc-Fc interaction of the antibody. The invention further provides pharmaceutical compositions comprising the antibodies and use of the antibodies for therapeutic and diagnostic procedures, in particular in cancer therapy.

RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application Serial No.17/929,799, filed Sep. 6, 2022, which claims priority to European PatentApplication Nos. 22173126.8, filed May 12, 2022, and 21195118.1, filedSep. 6, 2021, the entire disclosures of which are hereby incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Sep. 6, 2022, isnamed 734342_GMB9-019CON_ST26.xml and is 50,729 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies capable of binding to CD27and to antibody variants thereof comprising one or more mutations in theFc region and to the use of such antibodies and Fc variants.

BACKGROUND OF THE INVENTION

CD27 (TNFRSF7) is a 55 kDa type I transmembrane protein member of thetumor necrosis factor (TNF) receptor superfamily (TNFRSF) whichco-stimulates T-cell activation after binding to its ligand CD70. It isexpressed in humans on the cell membrane of T, B, NK cells, and theirimmediate precursors, all of them part of the lymphoid lineage. On humanT cells, CD27 is expressed on resting αβ CD4⁺ (Treg and conventional Tcells), CD8⁺ T cells, stem-cell memory cells, and central-memory-likecells. On human B cells, CD27 is a memory B cell marker and CD27signaling promotes differentiation of B cells into plasma cells.

The only known ligand for CD27 is the type II transmembrane protein CD70(Tumor Necrosis Factor Superfamily member 7, TNFSF7; CD27 ligand,CD27L), which is quite restrictively and only transiently expressed onactivated immune cells, including T, B, NK, and dendritic cells (DCs).

Upon binding of CD27 to CD70, a truncated 32 kDa form of CD27 can bereleased (known as soluble CD27, sCD27) through the action of matrixmetalloproteinases.

CD27 plays a role in early generation of a primary immune response andis required for generation and long-term maintenance of T cell immunity.CD27-CD70 binding leads to activation of NF-KB and MAPK8/JNK pathways.Adaptor proteins TRAF2 and TRAF5 have been shown to mediate thesignaling resulting from CD27 engagement.

To unlock their effector functions, T cells require T-cell antigenreceptor-mediated recognition of their cognate antigen in the context ofmajor histocompatibility complex (MHC) molecules on the surface ofantigen presenting cells (APCs), and activation of costimulatoryreceptors. CD27 and CD28 are considered the most important costimulatoryreceptors expressed on T cells.

In mice, CD27 stimulation during the priming phase of T-cell activation,has been found to promote clonal expansion of antigen-specific CD4⁺ andCD8⁺ T cells by IL-2-independent survival signaling (Carr JM et al, ProcNatl Acad Sci USA 2006 Dec 19; 130(51):19454-9). CD27 also counteractsapoptosis of activated T cells throughout successive divisions and wasalso shown to play an important role in memory differentiation of mouseCD8⁺ T cells. (van de Ven K, Borst J. Immunotherapy 2015;7(6):655-67).As a result, CD27 stimulation promotes the generation of effector Tcells in lymphoid organs and broadens the responder T-cell repertoire.In human naïve T cells, CD27 stimulation promotes T helper-1 (Th1)differentiation of CD4⁺ T cells and supports effector differentiation ofcytotoxic T-lymphocytes (Oosterwijk et al, Int Immunol. 2007 Jun;19(6):713-8).

Contrarily to its presence on tumor cells in some hematologicalmalignancies, CD27 expression has not been detected on tumor cells insolid malignancies. However, CD27-expressing lymphoid cells have beendescribed in the tumor microenvironment of both hematologicalmalignancies and solid cancers.

In the treatment of cancer, engagement and stimulation of the immuneresponse has been shown to induce and/or enhance anti-tumor immunityresulting in clinical responses, as exemplified by the clinical successof immune checkpoint inhibitors. An active immune response and/orexisting anti-tumor immunity can be increased by providingco-stimulatory signaling, for example CD27 co-stimulatory signaling.

In mouse tumor models, T-cell functions and therefore antitumor immunitycan be enhanced by agonistic CD27 antibodies. In hCD27-transgeniclymphoma mouse models, CD27 activation using agonistic antibodies showedpotent antitumor activity and induction of protective immunity, which isdependent on CD4⁺ and CD8⁺ T cells (He LZ et al., J Immunol. 2013 Oct15;191(8):4174-83). Furthermore, CD27 activation using monoclonalantibodies prevented tumor growth in mouse xenografts, including modelsderived from leukemia (Vitale et al, Keler T. Clin Cancer Res. 2012 Jul15;18(14):3812-21), melanoma (Roberts DJ, et al., J Immunother. 2010Oct;33(8):769-79), colon carcinoma, and thymoma (He LZ, et al., JImmunol. 2013 Oct 15;191(8):4174-83), among others.

Monoclonal IgG1 agonistic antibodies against human CD27 have beendisclosed in the prior art.

WO2008/051424 relates to CD27 agonists, preferably an agonistic CD27antibody, alone or in association with another moiety such as immunestimulant or immune modulator for treatment of cancer, infection,inflammation, allergy, and autoimmunity and for enhancing the efficacyof vaccines but does not disclose the sequence of any CD27 antibodies.

In WO2012/004367 a humanized anti-human CD27 agonistic antibody(designated hCD27.15) is described. It is reported that hCD27.15 doesnot require crosslinking by FcyR expressing cells to activateCD27-mediated co-stimulation of the immune response. However, thisantibody does not bind to a frequently occurring SNP in hCD27 (A59T) anddoes not bind to cynomolgus CD27.

WO2011/130434 discloses a human agonistic anti-human CD27 antibodydesignated 1F5, which activates CD27 upon crosslinking by FcyRexpressing cells and further is ligand (sCD70) blocking. 1F5 is reportedto have CDC and ADCC activity on target cells and to enhance the immuneresponse and to have anti-tumor activity in mouse models.

WO2018/058022 discloses the agonistic murine anti-human CD27 antibody131A and humanized versions thereof. It is disclosed that 131A binds thefrequent occurring SNP in hCD27 (A59T) and to cynomolgus CD27.WO2018/058022 further discloses that antibody 131A had greateranti-tumor response compared to the antibody 1F5 in a mouse tumor model.

WO2019/195452 discloses the non-ligand blocking agonistic anti-humanCD27 antibody designated BMS-986215 which is reported to have a higheraffinity for human and cynomolgus CD27 than the CD27-antibody 1F5mentioned above. It is disclosed that CD27 co-stimulation of T cells bybinding to its ligand CD70 occurs in the presence of BMS-986215. It isfurther disclosed that BMS-986215 reduces the suppression ofCD4⁺responder T cells by regulatory T-cells (Tregs) and that BMS-986215induces modest ADCC and low levels of ADCP, CDC and binds C1q. It isfurther disclosed that BMS-986215 only has weak agonist activity in theabsence of FcyR and in the absence of soluble CD70.

Anti-CD27 antibodies must induce clustering of CD27 on the plasmamembrane to induce CD27 agonism. In the case of wild type IgG1antibodies, clustering of CD27 may be achieved through interaction ofmembrane-bound CD27 antibodies with FcyR-bearing cells, such asmonocytes, macrophages, B cells and other immune cells. As aconsequence, anti-CD27 IgG1 molecules may be less efficient when thenumber of FcyR-expressing cells is limited. In addition, FcyR engagementmay also result in undesired effector functions, such as activation ofantibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), and complement-dependent cytotoxicity(CDC), which may cause unwanted depletion of CD27-positive T cells.

Optimization of the effector functions by modifications of the Fc regionof the antibody may improve the effectivity of therapeutic antibodiesfor treating cancer or other diseases, e.g., to improve the ability ofan antibody to elicit an immune response to antigen-expressing cells.Such efforts are described in, e.g., WO 2013/004842 A2; WO 2014/108198A1; WO2018/146317; WO2018/083126; WO 2018/031258 A1; Dall’Acqua, Cook etal. J Immunol 2006, 177(2): 1129-1138; Moore, Chen et al. MAbs 20102(2): 181-189; Desjarlais and Lazar, Exp Cell Res 2011, 317(9):1278-1285; Kaneko and Niwa, BioDrugs 2011, 25(1): 1-11; Song, Myojo etal., Antiviral Res 2014, 111: 60-68; Brezski and Georgiou, Curr OpinImmunol 2016, 40: 62-69; Sondermann and Szymkowski, Curr Opin Immunol2016, 40: 78-87; Zhang, Armstrong et al. MAbs 2017, 9(7): 1129-1142.;Wang, Mathieu et al. Protein & Cell 2018, 9(1): 63-73; Beurskens FJ etal., Science. 2014 Mar 14;343(6176):1260-3).

Despite these and other efforts in the art, however, there is a need foragonistic CD27 therapeutic antibodies with increased agonism and/orincreased potency and/or which are efficacious even when the number ofFcyR-expressing cells is limited. It is thus an object of the presentinvention to provide an anti-CD27 antibody having high potency andagonism and which induces higher activation of T-cell proliferationindependently of the number of FcgR-expressing cells providing thesecondary crosslinking for the clustering of CD27 on cell membrane.Thus, it is a further object to provide an anti-CD27 antibody which doesnot require crosslinking by FcyR expressing cells to activateCD27-mediated co-stimulation of the immune response. It is a furtherobject to provide an anti-CD27 antibody which binds to human CD27 andwhich further binds to the frequently occurring SNP in hCD27 (A59T) andwhich also binds to cynomolgus CD27. It is a further object of thepresent invention to provide CD27 agonist antibodies that induce CD27agonism through enhanced IgG hexamer formation, independent of secondarycrosslinking by C1q or in an FcyR-independent manner. In the context ofcancer, such antibodies may increase antitumor immunity. There remains aneed for anti-CD27 antibodies exhibiting potent agonistic activitiesthat enhance antitumor immune responses.

SUMMARY OF THE INVENTION

The present invention concerns CD27 binding antibodies and Fc variantsthereof.

So, in one aspect, the invention relates to an antibody comprising atleast one antigen-binding region capable of binding to human CD27wherein said antibody comprises a heavy chain variable (VH) region CDR1,CDR2, and CDR3 comprising the sequences as set forth in SEQ ID NOs: 5,6, and 7, respectively, and a light chain variable (VL) region CDR1,CDR2, and CDR3 comprising the sequences as set forth in SEQ ID NO: 9, 10and 11, respectively.

In one aspect the invention relates to an antibody comprising the VH andVL regions comprising the sequences as set forth in SEQ ID NO: 4 and SEQID NO: 8, respectively.

In one aspect the invention relates to an antibody comprising the VH andVL regions comprising the sequences as set forth in SEQ ID NO: 4 and SEQID NO: 8, respectively and further comprising a light chain constantregion (CL) and a heavy chain constant region (CH).

In one aspect the invention relates to an antibody comprising the VH andVL regions comprising the sequences as set forth in SEQ ID NO: 4 and SEQID NO: 8, respectively and further comprising a light chain constantregion (CL) and a heavy chain constant region (CH) wherein the antibodyis of the human IgG1 isotype.

In one aspect the invention relates to an antibody as described abovewhich has a modified Fc region wherein the amino acid residue at theposition corresponding to position E345 or E430 in a human IgG1 heavychain according to Eu numbering is selected from the group comprising:A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, Wand Y.

In one aspect the invention relates to any of the antibodies asdescribed above and which further has a modified Fc region wherein theamino acid residue at the position corresponding to position P329 in ahuman IgG1 heavy chain according to Eu numbering is R.

In one aspect the invention relates to an antibody comprising a heavychain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequencesas set forth in SEQ ID NOs: 5, 6, and 7, respectively, and a light chainvariable (VL) region CDR1, CDR2, and CDR3 comprising the sequences asset forth in SEQ ID NO: 9, 10 and 11, respectively and furthercomprising a modified Fc region wherein the amino acid residue at thepositions corresponding to position E345 and P329 in a human IgG1 heavychain according to Eu numbering are both R.

In one aspect the invention relates to a human or a humanized antibody.

In one aspect the invention relates to an antibody comprising:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4;-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8;-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 15; and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In one aspect the invention relates to an antibody comprising:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4;-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8;-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 15; and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In one aspect the invention relates to an antibody comprising a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 35 anda light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

In one aspect, the invention relates to an isolated nucleic acidencoding the antibody according to any aspect or embodiment herein.

In one aspect, the invention relates to an expression vector comprisingsuch a nucleic acid.

In one aspect, the invention relates to a recombinant host cell whichproduces an antibody according to any aspect or embodiment herein.

In one aspect, the invention relates to a method of producing anantibody according to any aspect or embodiment herein, comprisingcultivating such a recombinant host cell in a culture medium and underconditions suitable for producing the antibody.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an antibody as defined in any aspect or embodiment herein,and a pharmaceutically acceptable carrier.

In one aspect, the invention relates to an antibody according to anyaspect or embodiment herein for use as a medicament.

In one aspect, the invention relates to an antibody according to anyaspect or embodiment herein for use in treating or preventing cancer.

In one aspect the invention relates to a method of treating a disease,the method comprising administering an antibody according to any aspector embodiment herein, a composition according to any aspect orembodiment herein, or a pharmaceutical composition according to anyaspect or embodiment herein, to a subject in need thereof.

In one aspect the invention relates to a kit-of-parts, such as a kit foruse as a companion diagnostic/for identifying within a population ofpatients those patients which have a propensity to respond to treatmentwith an antibody according to any aspect or embodiment herein.

In one aspect the invention relates to an anti-idiotypic antibody, whichbinds to the antigen-binding region capable of binding to CD27 asdefined in any one aspect or embodiment herein.

LEGENDS TO THE FIGURES

FIG. 1 shows CD27 agonist activity of anti-CD27 antibodies andhexamerization-enhanced Fc variants thereof as determined in a CD27Jurkat Reporter BioAssay. Thaw-and-Use GloResponse NF_(K)B-luc2/CD27Jurkat reporter cells were incubated for 6 h with antibody concentrationseries (from left to right: 0.04 µg/mL, 0.30 µg/mL, 2.50 µg/mL, and 20µg/mL) of the indicated antibodies. Luciferase activity, as a readoutfor CD27 intracellular signaling, was quantified by determining theluminescence (RLU: relative luminescence units). The followingantibodies were included as WT IgG1 and/or variants with an E430G orE345R mutation, as indicated: non-binding anti-HIV-gp120 controlantibody comprising the E345R mutation (IgG1-b12-E345R, ctrl), anti-CD27antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D,IgG1-CD27-E, and IgG1-CD27-F, and prior art anti-CD27 benchmarkantibodies IgG1-CD27-131A and IgG1-CD27-15.

FIG. 2 shows binding of anti-CD27 antibodies to (A,B) human and (C,D)cynomolgus monkey CD27 expressed on (A,C) T cells in PBMC or (B,D)CD27-transfected HEK293F cells, as determined by flow cytometry.Antibody binding is presented as the median fluorescence intensity(MFI). The anti-HIV-gp120 antibody IgG1-b12-FEAR (ctrl) was included asnon-binding negative control antibody.

FIG. 3 shows binding of anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B,and IgG1-CD27-C to human CD27-A59T variant expressed on HEK293F cells,as determined by flow cytometry. Antibody binding is presented as themedian MFI. The anti-HIV-gp120 antibody IgG1-b12-FEAL (ctrl) wasincluded as non-binding negative control antibody.

FIG. 4 shows heatmaps of the proliferation of TCR stimulated (A) CD8⁺and (B) CD4⁺ T cells in the presence of 1 µg/mL CD27-specific antibodyvariants IgG1-CD27-A, -B, or -C harboring the Fc mutations E430R orE345R in combination with the Fc mutations P329R, G237A, or K326A-E33A,as determined by flow cytometry in a CSFE dilution assay. PMBC from fourhuman healthy donors were used as a source of T cells. T-cellproliferation was expressed as the T-cell division index or thepercentage of proliferated T cells, that was calculated by gating forthe cells that have gone through CFSE dilution (CFSE^(low) ^(peaks)) byusing the FlowJo software.

FIG. 5 shows the (A-D) percentage of proliferated T cells, (E, F) theexpansion index of (A, B) unstimulated or (C-F) TCR stimulated (A, C, E)CD4⁺ or (B, D, F) CD8⁺ T cells after incubation of human healthy donorPBMC with IgG1-CD27-A, IgG1-CD27-A-P329R-E345R or prior art anti-CD27clones IgG1-CD27-131A, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215, asdetermined by flow cytometry. The anti-HIV-gp120 antibody variantIgG1-b12-E345R-P329R (ctrl) was included as non-binding negative controlantibody. % Proliferated cells were calculated by gating for the cellsthat have gone through CFSE dilution (CFSE^(low) ^(peaks)). Expansionindex identifies the fold increase of cells in the wells and wascalculated using the Proliferation Modeling tool in FlowJo version 10.Manual adjustments to the peaks were made where necessary to define thenumber of the peaks present more consistently.

FIG. 6 shows binding of C1q to membrane-bound CD27 antibodies of theinvention, as determined by FACS. IgG1-CD27-A variants containing aE430G or E345R hexamerization-enhancing mutation (IgG1-CD27-A-E430G andIgG1-CD27-A-E345R) and the P329R mutation (IgG1-CD27-A-P329R-E345R) weretested for their capacity to bind to C1q. The anti-HIV-gp120 antibodyIgG1-b12-F405L (ctrl) was included as non-binding negative controlantibody.

FIG. 7 shows binding of IgG1-CD27-A-P329R-E345R to human Fc receptors asdetermined by surface plasmon resonance (SPR). Biacore surface chipswere covalently linked with anti-His antibody and coated withrecombinant His-tagged Fc receptors (A) FcyRla, (B) FcyRlla-H, (C)FcyRlla-R, (D) FcyRllb, (E) FcyRllla-F, or (F) FcyRllla-V. Theanti-HIV-gp120 antibody IgG1-b12 (ctrl) was included as a reference.Shown are absolute resonance units as determined by Biacore SPR afterbackground subtraction (no Fc receptor flow-cell).

FIG. 8 shows binding of IgG1-CD27-A-P329R-E345R to human (A) CD4⁺ and(B) CD8⁺ T-cell subsets in human healthy donor PBMC samples, asdetermined by flow cytometry. Negative control antibodyIgG1-b12-P329R-E345R (ctrl) is an anti-HIV gp120 non-binding isotypecontrol antibody comprising the P329R and E345R mutations. Datapresented is the mean MFI +/- SD of duplicate samples.

FIG. 9 shows CD27 agonist activity of anti-CD27 antibodies in presenceand absence of FcYR-mediated crosslinking, as determined in a reporterassay. A fixed number of NF_(K)B-luc2/CD27 Jurkat reporter cells wascultured with (A-E) IgG1-CD27-A-P329R-E345R or IgG1-CD27-A, (F-J)IgG1-CD27-131A, IgG1-CD27-CDX1127 or IgG1-CD27-BMS986215, in (A,F)absence or (B-J) presence of FcyRllb-CHO-K1 cells, at aNF_(K)B-luc2/CD27 Jurkat : FcyRllb CHO-K1 ratio of (B,G) 1:1, (C,H) 1:⅓,(D,I) 1:⅑, or (E,J) 1:1/27. IgG1-b12-P329R-E345R and IgG1-b12 areanti-HIV gp120 non-binding control antibodies (ctrl). Luminescence wasmeasured as a readout for CD27 activation and presented as relativeluminescence units (RLU).

FIG. 10 shows the human IgG levels in plasma of SCID mice, afterintravenous injection of 25 mg/kg IgG-CD27-A or IgG-CD27-A-P329R-E345Rantibodies. Total human IgG plasma concentrations were determined bysandwich ELISA and plotted against time after injection. Data shown aremean plasma concentrations +/- SEM of blood samples per group (n=3mice).

FIG. 11 shows the percentage of viable CD27⁺ Daudi cells afterco-culturing for 4 h with hMDM (E:T = 2:1) in the presence ofIgG1-CD27-A-P329R-E345R or wild-type CD20 antibody IgG1-CD20. Daudicells were labeled with CellTrace™ Violet and cell viability wasmeasured by flow cytometry. Data shown are the mean of duplicates ± SDpercentage of viable Daudi cells (TO-PRO-3⁻CTV⁺CD11b⁻) normalized to theno antibody controls for one donor out of four tested in twoexperiments.

FIG. 12 shows C4d deposition upon incubation of IgG1-CD27-A-P329R-E345Rin NHS as determined by ELISA. IgG1-b12-P329R-E345R is an isotypecontrol antibody and IgG1-b12 is a control antibody with a WT Fc domain;IgG1-b12-RGY is a positive control antibody for C4d deposition(hexameric antibody in solution). Shown is mean ± SD of triplicates ofone representative experiment out of three performed.

FIG. 13 shows the inhibition of CD70 binding on Daudi cells by anti-CD27antibodies. CD27⁺ Daudi cells were incubated with 6 µg/mL biotinylatedrecombinant human CD70 ECD in the presence or absence of 50 µg/mL of thenon-binding control antibodies (IgG1-b12-P329E-E345R or IgG1-b12) orCD27 antibodies (IgG1-CD27-A, IgG1-CD27-A-P329R-E345R,IgG1-CD27-CDX1127, IgG1-CD27-BMS986215, or IgG1-CD27-131A). Binding ofthe biotinylated CD70 fragment to the Daudi cells was detected by flowcytometry using BV421-labeled streptavidin. Data shown are the gMFI ± SDfrom duplicate wells of one representative experiment out of threeperformed.

FIG. 14 shows expression levels of T-cell activation markers inpolyclonally activated CD4⁺ and CD8⁺ T cells upon treatment withanti-CD27 antibodies. Human healthy donor PBMC were incubated with 0.1µg/mL CD3 antibody and 30 µg/mL of IgG1-CD27-A-P329R-E345R, CD27antibody benchmarks or non-binding control antibody IgG1-b12-P329R-E345Rfor two or five days. The expression levels of T-cell activation markersHLA-DR, CD69, GITR, CD25, CD107a, and 4-1BB on the surface of (A) CD4⁺and (B) CD8⁺ T cells in antibody-treated samples were quantified by flowcytometry and presented as mean fold change in MFI (± SD) relative tothe nonbinding control sample of the same donor. Dotted lines indicatethe fold change for cells treated with IgG1-b12-P329R-E345R, which wasused as a nonbinding control and set to 1. Data shown are from threedonors tested in duplicate in one experiment.

FIG. 15 shows percentages of OVA-specific CD8⁺ T cells in spleen ofhCD27-KI mice after immunization with OVA and treatment with anti-CD27antibodies. hCD27-KI mice were injected s.c. with 5 mg OVA on days 0, 12and 21, and simultaneously treated i.v. with 30 mg/kgIgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127 or non-binding controlantibody IgG1-b12-P329R-E345R. On day 28, mice were euthanized, spleenswere resected, and processed as single cell suspensions. Expansion ofOVA specific CD8⁺ T cells was evaluated by flow cytometry. Data shownare the mean of % OVA⁺ of CD8⁺ cells ± SD per treatment group (5 miceper group) from one experiment performed.

FIG. 16 shows the number of IFNy-producing splenocytes on day 28 afterimmunization with OVA and treatment with anti-CD27 antibodies asmeasured by IFNγ-ELISpot. hCD27-KI mice were injected s.c. with 5 mg OVAon days 0, 12 and 21, and simultaneously treated i.v. with 30 mg/kgIgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127, or non-binding controlantibody IgG1-b12-P329R-E345R. On day 28, spleens were resected,processed as single cell suspensions and IFNy-producing splenocytes weredetected using IFNy-ELISpot. Data shown are the mean number of spots perwell ± SEM of each treatment group from one experiment performed (5 miceper group).

FIG. 17 shows the percentage of activated CD8⁺ T cells in the spleen ofhCD27-KI mice after immunization with OVA and treatment with anti-CD27antibodies. hCD27-KI mice were injected s.c. with 5 mg OVA on days 0, 12and 21, and simultaneously treated i.v. with 30 mg/kgIgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127, or non-binding controlantibody IgG1-b12-P329R-E345R. On day 28, mice were euthanized, spleenswere resected, and processed as single cell suspensions. Activation ofCD8⁺ T cells was evaluated in spleen samples by measuring the percentagePD-1⁺ of CD8⁺ cells in spleen by flow cytometry. Data shown are the mean± SD per treatment group (5 mice per group) from one experimentperformed.

FIG. 18 shows percentages of effector CD8⁺ T cells in the spleen ofhCD27-KI mice after immunization with OVA and treatment with anti-CD27antibodies. hCD27-KI mice were injected s.c. with 5 mg OVA on days 0, 12and 21, and simultaneously treated i.v. with 30 mg/kgIgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127, or non-binding controlantibody IgG1-b12-P329R-E345R. On day 28 mice, were euthanized, spleenswere resected, and processed as single cell suspensions. Expansion ofmemory T cells was evaluated by expression of CD44 and CD62L by flowcytometry. Data shown are the mean ± SD per treatment group (5 mice pergroup) from one experiment performed. (A) PercentageCD8⁺CD44⁺CD62L-effector memory of CD45⁺ cells. (B) PercentageCD44⁺CD62L⁻ effector memory of CD8⁺ T cells. (C) PercentageCD8⁺CD44⁻CD62L⁻pre-effector of CD45⁺cells. (D) PercentageCD44⁻CD62L⁻pre-effector of CD8⁺ T cells.

FIG. 19 shows percentage of T cells in the spleen of hCD27-KI mice afterimmunization with OVA and treatment with anti-CD27 antibodies. hCD27-KImice were injected s.c. with 5 mg OVA on days 0, 12 and 21, andsimultaneously treated i.v. with 30 mg/kg IgG1-CD27-A-P329R-E345R,IgG1-CD27-CDX1127, or non-binding control antibody IgG1-b12-P329R-E345R.On day 28, mice were euthanized, spleens were resected, and processed assingle cell suspensions. CD3⁺ cells in the blood and spleens wereevaluated by flow cytometry. Data shown are the mean ± SD per treatmentgroup (5 mice per group) from one experiment performed.

FIG. 20 shows the effect of IgG1-CD27-A-P329R-E345R on T-cell cytokineproduction in antigen-specific studies. Cocultures ofCLDN6-TCR-expressing CD8+ T cells that (A) express endogenous PD-1 or(B) overexpress PD-1 and autologous CLDN6-expressing iDC were incubatedwith 10 µg/mL IgG1-CD27-A-P329R-E345R, CD27 benchmark antibodyIgG1-CD27-131A, or nonbinding control antibody IgG1-b12-P329R-E345R fortwo days. Cytokine levels in coculture supernatants were analyzed bymultiplex ECLIA. Data shown are mean concentrations ± SD of triplicatewells from one representative donor out of seven tested in twoexperiments performed. Abbreviations: CLDN6 = claudin 6; ECLIA =electrochemiluminescence assay; iDC = immature dendritic cell; PD-1 =programmed cell death protein 1; SD = standard deviation; TCR = T cellreceptor.

FIG. 21 shows expression of cytotoxicity-associated molecules inantigen-specific CD8+T cells incubated with IgG1-CD27-A-P329R-E345R.CLDN6-TCR-electroporated CD8+ T cells were cocultured withhCLDN6-MDA-MB-231 cells in the presence of IgG1-CD27-A-P329R-E345R, CD27benchmark IgG1-CD27-131A, or nonbinding control antibodyIgG1-b12-P329R-E345R for two days. Intracellular expression of GzmB andCD107a was determined by flow cytometry. The percentage of CD8+ T cellsexpressing both GzmB and CD107a, as well as expression levels of GzmBand CD107a (MFI normalized to IgG1-b12-P329R-E345R) in CD8+ T cells isshown. Data shown are mean ± SD of six donors tested in single replicatein experiments two experiments. **, P<0.01; *, P<0.05; Friedman-testwith Dunn’s multiple comparisons test. Abbreviations: CLDN6 = claudin 6;GzmB = granzyme B; MFI = mean fluorescence intensity; SD = standarddeviation; TCR = T-cell receptor.

FIG. 22 shows antigen-specific CD8+ T-cell mediated tumor cell kill inthe presence of IgG1-CD27-A-P329R-E345R. CD8+ T-cell mediated kill ofhCLDN6-MDA-MB-231 cells was evaluated by real-time cell analysis. CLDN6TCR electroporated CD8+ T cells were cocultured with hCLDN6-MDA-MB-231cells in the presence of IgG1-CD27-A-P329R-E345R, CD27 benchmarkIgG1-CD27-131A, or nonbinding control antibody IgG1-b12-P329R-E345R forfive days. Cell index values were derived from impedance measurementsconducted at two-hour intervals. AUC were obtained from cell index dataover five days of coculture. The AUC of each treatment condition wasnormalized to IgG1-b12-P329R-E345R-treated cultures from the same donor.Data shown are mean ± SD from six donors tested in duplicate inexperiments in two experiments. **, P<0.01; Friedman-test with Dunn’smultiple comparisons test. Abbreviations: AUC = area under the curve;CLDN6 = claudin 6; SD = standard deviation; TCR = T-cell receptor.

FIG. 23 shows absolute cell numbers of CD4+ and CD8+ T cells and NKcells in primary tumor cultures after treatment withIgG1-CD27-A-P329R-E345R. Human NSCLC tumor tissues were cultured withlow-dose IL-2 (45 to 50 U/mL) in the presence or absence of 10 µg/mLIgG1-CD27-A-P329R-E345R. Absolute cell counts of the TIL subsets weredetermined by flow cytometry after 14 days of treatment. Data shown areaverage ± SD of four replicate wells from one out of five tumor tissuestested in one experiment out of four performed. Abbreviations: IL =interleukin; NK = natural killer; NSCLC = non-small cell lung cancer; SD= standard deviation; U/mL = units per mL.

FIG. 24 shows molecular proximity determined by bioluminescenceresonance energy transfer (BRET) analysis betweenIgG1-CD27-A-P329R-E345R antibodies on the cell surface of Daudi andhuCD27-K562 cells. Cells were incubated with mixtures of NanoLuc-(donor) and HaloTag- (acceptor) tagged antibodies (5 µg/mL each):IgG1-CD27-A-P329R-E345R, WT IgG1-CD27-A or nonbinding controlIgG1-b12-P329RE345R as indicated. The antibody pairIgG1-CD20-11B8-E430G-LNLuc and IgG1-CD37-37.3-E430G-LHalo was used aspositive control. BRET was calculated in milliBRET units (mBU) = (618nm_(em)/460 nm_(em)) × 1000, and corrected for donor bleed-through bysubtracting no-ligand control values. Data shown are the corrected BRETfrom duplicate wells of one representative experiment out of threeperformed.

FIG. 25 shows binding of IgG1-CD27-A-P329R-E345R to M0 and M1macrophages compared to a WT IgG1 antibody (IgG1-b12) with an irrelevantantigen-binding region as a positive control for FcyRla binding, and avariant of the same antibody carrying the P329R and E345R mutations(IgG1-b12-P329R-E345R). Binding of the antibodies to the macrophages wasdetected by flow cytometry using PE-labeled goat anti-human secondaryantibody. Data shown are mean + SD of two donors tested.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen. The antibody of the present invention comprises anFc-domain of an immunoglobulin and an antigen-binding region. Anantibody generally contains two CH2-CH3 regions and a connecting region,e.g., a hinge region, e.g. at least an Fc-domain. Thus, the antibody ofthe present invention may comprise an Fc region and an antigen-bindingregion. The variable regions of the heavy and light chains of theimmunoglobulin molecule contain a binding domain that interacts with anantigen. The constant or “Fc” regions of the antibodies may mediate thebinding of the immunoglobulin to host tissues or factors, includingvarious cells of the immune system (such as effector cells) andcomponents of the complement system such as C1q, the first component inthe classical pathway of complement activation. As used herein, unlesscontradicted by context, the Fc region of an immunoglobulin typicallycontains at least a CH2 domain and a CH3 domain of an immunoglobulin CH,and may comprise a connecting region, e.g., a hinge region. An Fc-regionis typically in dimerized form via, e.g., disulfide bridges connectingthe two hinge regions and/or non-covalent interactions between the twoCH3 regions. The dimer may be a homodimer (where the two Fc regionmonomer amino acid sequences are identical) or a heterodimer (where thetwo Fc region monomer amino acid sequences differ in one or more aminoacids). An Fc region-fragment of a full-length antibody can, forexample, be generated by digestion of the full-length antibody withpapain, as is well-known in the art. An antibody as defined herein may,in addition to an Fc region and an antigen-binding region, furthercomprise one or both of an immunoglobulin CH1 region and a CL region. Anantibody may also be a multi-specific antibody, such as a bispecificantibody or similar molecule. The term “bispecific antibody” refers toan antibody having specificities for at least two different, typicallynon-overlapping, epitopes. Such epitopes may be on the same or differenttargets. If the epitopes are on different targets, such targets may beon the same cell or different cells or cell types. As indicated above,unless otherwise stated or clearly contradicted by the context, the termantibody herein includes fragments of an antibody which comprise atleast a portion of an Fc-region and which retain the ability tospecifically bind to the antigen. Such fragments may be provided by anyknown technique, such as enzymatic cleavage, peptide synthesis andrecombinant expression techniques. It has been shown that theantigen-binding function of an antibody may be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “Ab” or “antibody” include, without limitation, monovalentantibodies (described in WO2007059782 by Genmab); heavy-chainantibodies, consisting only of two heavy chains and naturally occurringin e.g. camelids (e.g., Hamers-Casterman (1993) Nature 363:446);ThioMabs, Roche, WO2011069104); strand-exchange engineered domain (SEEDor Seed-body) which are asymmetric and bispecific antibody-likemolecules (Merck, WO2007110205); Triomab (Pharma/Fresenius Biotech,Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp(Regeneron, WO2010151792); Azymetric Scaffold (Zymeworks/Merck,WO2012/058768); mAb-Fv (Xencor, WO2011/028952); Xmab (Xencor); Dualvariable domain immunoglobulin (Abbott, DVD-Ig,U.S. Pat. No. 7,612,181);Dual domain double head antibodies (Unilever; Sanofi Aventis,WO20100226923); Di-diabody (ImClone/Eli Lilly); Knobs-into-holesantibody formats (Genentech, WO9850431 ); DuoBody (Genmab, WO2011/131746); Bispecific IgG1 and IgG2 (Pfizer/Rinat, WO11143545);DuetMab (Medlmmune, US2014/0348839); Electrostatic steering antibodyformats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133;Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciencesCorporation, WO11143545); CrossMAbs (Roche, WO2011117329); LUZ-Y(Genentech); Biclonic (Merus, WO2013157953); Dual Targeting domainantibodies (GSK/Domantis); Two-in-one Antibodies or Dual action Fabsrecognizing two targets (Genentech, Novlmmune, Adimab); Cross-linkedMabs (Karmanos Cancer Center); covalently fused mAbs (AIMM); CovX-body(CovX/Pfizer); FynomAbs (Covagen/Janssen ilag); DutaMab (Dutalys/Roche);iMab (Medlmmune); IgG-like Bispecific (ImClone/Eli Lilly, Shen, J., etal. J Immunol Methods, 2007. 318(1-2): p. 65-74); TIG-body, DIG-body andPIG-body (Pharmabcine); Dual-affinity retargeting molecules (Fc-DART orIg-DART, Macrogenics, WO/2008/157379, WO/2010/080538); BEAT (Glenmark);Zybodies (Zyngenia); approaches with common light chain (Crucell/ Merus,US7262028) or common heavy chains (kλBodies by Novlmmune, WO2012023053),as well as fusion proteins comprising a polypeptide sequence fused to anantibody fragment containing an Fc-region like scFv-fusions, like BsAbby ZymoGenetics/BMS, HERCULES by Biogen Idec (US007951918); SCORPIONS(Emergent BioSolutions/Trubion and Zymogenetics/BMS); Ts2Ab(MedImmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92);scFv fusion (Genentech/Roche); scFv fusion (Novartis); scFv fusion(Immunomedics); scFv fusion (Changzhou Adam Biotech Inc, CN 102250246);TvAb (Roche,WO 2012025525, WO 2012025530); mAb2 (f-Star, WO2008/003116);and dual scFv-fusion. It should be understood that the term antibody,unless otherwise specified, includes monoclonal antibodies (such ashuman monoclonal antibodies), polyclonal antibodies, chimericantibodies, humanized antibodies, monospecific antibodies (such asbivalent monospecific antibodies), bispecific antibodies, antibodies ofany isotype and/or allotype; antibody mixtures (recombinant polyclonals)for instance generated by technologies exploited by Symphogen and Merus(Oligoclonics), multimeric Fc proteins as described in WO2015/158867,and fusion proteins as described in WO2014/031646. While these differentantibody fragments and formats are generally included within the meaningof antibody, they collectively and each independently are uniquefeatures of the present invention, exhibiting different biologicalproperties and utility.

An “agonistic antibody” for a natural receptor is a compound which bindsthe receptor to form a receptor-antibody complex and which activatessaid receptor, thereby initiating a pathway signaling and furtherbiological process.

The term “agonism” and “agonistic” are used interchangeably herein andrefer to or describe an antibody which is capable of, directly orindirectly, substantially inducing, promoting, or enhancing CD27biological activity or activation. Optionally, an “agonistic CD27antibody” is an antibody which is capable of activating CD27 receptor bya similar mechanism as the ligand for CD27, known as CD70 (TumorNecrosis Factor Superfamily member 7, TNFSF7; CD27 ligand, CD27L), whichresults in an activation of one or more intracellular signaling pathwaywhich may include activation of NF-KB and MAPK8/JNK pathways. “Agonism”as defined herein may be determined according to Example 2 herein.

A “CD27 antibody” or “anti-CD27 antibody” as described herein is anantibody which binds specifically to the protein CD27, in particular tohuman CD27.

A “variant” as used herein refers to a protein or polypeptide sequencewhich differs in one or more amino acid residues from a parent orreference sequence. A variant may, for example, have a sequence identityof at least 80%, such as 90%, or 95%, or 97%, or 98%, or 99%, to aparent or reference sequence. Also, or alternatively, a variant maydiffer from the parent or reference sequence by 12 or less, such as 11,10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation(s) such as substitutions,insertions, or deletions of amino acid residues. Accordingly, a “variantantibody” or an “antibody variant”, used interchangeably herein, refersto an antibody that differs in one or more amino acid residues ascompared to a parent or reference antibody, e.g., in the antigen-bindingregion, Fc-region or both. Likewise, a “variant Fc region” or “Fc regionvariant” refers to an Fc region that differs in one or more amino acidresidues as compared to a parent or reference Fc region, optionallydiffering from the parent or reference Fc region amino acid sequence by12 or less, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation(s) suchas substitutions, insertions, or deletions of amino acid residues. Theparent or reference Fc region is typically the Fc region of a humanwild-type antibody which, depending on the context, may be a particularisotype. A variant Fc region may, in dimerized form, be a homodimer orheterodimer, e.g., where one of the amino acid sequences of thedimerized Fc region comprises a mutation while the other is identical toa parent or reference wild-type amino acid sequence. Examples ofwild-type (typically a parent or reference sequence) IgG CH and variantIgG constant region amino acid sequences, which comprise Fc region aminoacid sequences, are set out in Table 3.

The term “immunoglobulin heavy chain” or “heavy chain of animmunoglobulin” as used herein is intended to refer to one of the heavychains of an immunoglobulin. A heavy chain is typically comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region (abbreviated herein as CH) which defines the isotype ofthe immunoglobulin. The heavy chain constant region typically iscomprised of three domains, CH1, CH2, and CH3. The term “immunoglobulin”as used herein is intended to refer to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four potentially inter-connected by disulfide bonds. The structureof immunoglobulins has been well characterized (see for instanceFundamental Immunology Ch. 7 Paul, W., 2nd ed. Raven Press, N.Y. 1989).Within the structure of the immunoglobulin, the two heavy chains areinter-connected via disulfide bonds in the so-called “hinge region”.Equally to the heavy chains, each light chain is typically comprised ofseveral regions; a light chain variable region (abbreviated herein asVL) and a light chain constant region. The light chain constant regiontypically is comprised of one domain, CL. Furthermore, the VH and VLregions may be further subdivided into regions of hypervariability (orhypervariable regions which may be hypervariable in sequence and/or formof structurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). Each VH and VL is typically composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. CDR sequences herein are defined according to IMGT (seeLefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999] andBrochet X. Nucl. Acids Res. 36, W503-508 (2008)).

When used herein, the terms “half molecule”, “Fab-arm” and “arm” referto one heavy chain-light chain pair. When a bispecific antibody isdescribed to comprise a half-molecule antibody “derived from” a firstantibody, and a half-molecule antibody “derived from” a second antibody,the term “derived from” indicates that the bispecific antibody wasgenerated by recombining, by any known method, said half-molecules fromeach of said first and second antibodies into the resulting bispecificantibody. In this context, “recombining” is not intended to be limitedby any particular method of recombining and thus includes all of themethods for producing bispecific antibodies described herein below,including for example recombining by “half-molecule exchange” alsodescribed in the art as “Fab-arm exchange” and the DuoBody® method, aswell as recombining at nucleic acid level and/or through co-expressionof two half-molecules in the same cells.

The term “antigen-binding region” or “binding region” or antigen-bindingdomain as used herein, refers to the region of an antibody which iscapable of binding to the antigen. This binding region is typicallydefined by the VH and VL domains of the antibody which may be furthersubdivided into regions of hypervariability (or hypervariable regionswhich may be hypervariable in sequence and/or form of structurallydefined loops), also termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FRs). The antigen can be any molecule, such as a polypeptide,e.g., present on a cell, bacterium, or virion. The terms“antigen-binding region” and “antigen-binding site” and “antigen-bindingdomain” may, unless contradicted by the context, be used interchangeablyin the context of the present invention.

The terms “antigen” and “target” may, unless contradicted by thecontext, be used interchangeably in the context of the presentinvention.

The term “binding” as used herein refers to the binding of an antibodyto a predetermined antigen or target, typically with a binding affinitycorresponding to a K_(D) of 1E⁶ M or less, e.g. 5E⁷ M or less, 1E⁷ M orless, such as 5E⁸ M or less, such as 1E⁸ M or less, such as 5E⁹ M orless, or such as 1E⁹ M or less, when determined by biolayerinterferometry using the antibody as the ligand and the antigen as theanalyte and binds to the predetermined antigen with an affinitycorresponding to a K_(D) that is at least ten-fold lower, such as atleast 100-fold lower, for instance at least 1,000-fold lower, such as atleast 10,000-fold lower, for instance at least 100,000-fold lower thanits affinity for binding to a non-specific antigen (e.g., BSA, casein)other than the predetermined antigen or a closely-related antigen.

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction, andis obtained by dividing k_(d) by k_(a).

The term “k_(d)” (sec⁻¹), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the k_(off) value or off-rate.

The term “k_(a)” (M⁻¹ x sec⁻¹), as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction.Said value is also referred to as the k_(on) value or on-rate.

The term “CD27” as used herein, refers to the human protein entitledCD27, also known as tumor necrosis factor receptor superfamily member 7(TNFRSF7). In the amino acid sequence shown in SEQ ID NO: 1 (Uniprot IDP26842), amino acid residues 1-19 are a signal peptide, and amino acidresidues 20-240 are the mature polypeptide. Unless contradicted bycontext, CD27 may also refer to variants of CD27, isoforms and orthologsthereof. A naturally occurring variant of human CD27 comprising a A59Tmutation is shown in SEQ ID NO: 2.

In cynomolgus monkey (Macaca fascicularis), the CD27 protein has theamino acid sequence shown in SEQ ID NO: 3 (GenbankXP_005569963). In the240 amino acid sequence shown in SEQ ID NO: 3, the signal peptide is notdefined.

The term “antibody binding region” refers to a region of the antigen,which comprises the epitope to which the antibody binds. An antibodybinding region may be determined by epitope binding using biolayerinterferometry, by alanine scan, or by shuffle assays (using antigenconstructs in which regions of the antigen are exchanged with that ofanother species and determining whether the antibody still binds to theantigen or not). The amino acids within the antibody binding region thatare involved in the interaction with the antibody may be determined byhydrogen/deuterium exchange mass spectrometry and by crystallography ofthe antibody bound to its antigen.

The term “epitope” means an antigenic determinant which is specificallybound by an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids, sugar side chains or a combinationthereof and usually have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. The epitope may comprise amino acid residues whichare directly involved in the binding, and other amino acid residues,which are not directly involved in the binding, such as amino acidresidues which are effectively blocked or covered by the antibody whenit is bound to the antigen (in other words, the amino acid residue iswithin or closely adjacent to the footprint of the specific antibody).

The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibodycomposition”, “mAb”, or the like, as used herein refer to a preparationof antibody molecules of single molecular composition. A monoclonalantibody composition displays a single binding specificity and affinityfor a particular epitope. Accordingly, the term “human monoclonalantibody” refers to antibodies displaying a single binding specificitywhich have variable and constant regions derived from human germlineimmunoglobulin sequences. The human monoclonal antibodies may beproduced by a hybridoma which includes a B cell obtained from atransgenic or trans-chromosomal non-human animal, such as a transgenicmouse or rat, having a genome comprising a human heavy chain transgeneand a light chain transgene, fused to an immortalized cell. Monoclonalantibodies may also be produced from recombinantly modified host cells,or systems that use cellular extracts supporting in vitro transcriptionand/or translation of nucleic acid sequences encoding the antibody.

The term “isotype” as used herein refers to the immunoglobulin class(for instance IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or anyallotypes thereof, such as IgG1m(za) and IgG1m(f)) that is encoded byheavy chain constant region genes. Further, each heavy chain isotype canbe combined with either a kappa (κ) or lambda (λ) light chain.

The term “full-length antibody” when used herein, indicates that theantibody is not a fragment, but contains all of the domains of theparticular isotype normally found for that isotype in nature, e.g., theVH, CH1, CH2, CH3, hinge, VL and CL domains for an IgG1 antibody. In afull-length variant antibody, the heavy and light chain constant andvariable domains may in particular contain amino acid substitutions thatimprove the functional properties of the antibody when compared to thefull-length parent or wild type antibody. A full-length antibodyaccording to the present invention may be produced by a methodcomprising the steps of (i) cloning the CDR sequences into a suitablevector comprising complete heavy chain sequences and complete lightchain sequence, and (ii) expressing the complete heavy and light chainsequences in suitable expression systems. It is within the knowledge ofthe skilled person to produce a full-length antibody when starting outfrom either CDR sequences or full variable region sequences. Thus, theskilled person would know how to generate a full-length antibodyaccording to the present invention.

The term “human antibody”, as used herein, is intended to includeantibodies comprising variable and framework regions derived from humangermline immunoglobulin sequences and a human immunoglobulin constantdomain. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations, insertions or deletions introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody”, as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another non-humanspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “humanized antibody” as used herein, refers to a geneticallyengineered non-human antibody, which contains human antibody constantdomains and non-human variable domains modified to contain a high levelof sequence homology to human variable domains. This can be achieved bygrafting of the six non-human antibody complementarity-determiningregions (CDRs), which together form the antigen binding site, onto ahomologous human acceptor framework region (FR) (see WO92/22653 andEP0629240). In order to fully reconstitute the binding affinity andspecificity of the parental antibody, the substitution of frameworkresidues from the parental antibody (i.e., the non-human antibody) intothe human framework regions (back-mutations) may be required. Structuralhomology modeling may help to identify the amino acid residues in theframework regions that are important for the binding properties of theantibody. Thus, a humanized antibody may comprise non-human CDRsequences, primarily human framework regions optionally comprising oneor more amino acid back-mutations to the non-human amino acid sequence,and fully human constant regions. Optionally, additional amino acidmodifications, which are not necessarily back-mutations, may be appliedto obtain a humanized antibody with preferred characteristics, such asaffinity and biochemical properties.

The term “Fc region” or “Fc domain” as used herein may be usedinterchangeably and refers to a region of the heavy chain constantregion comprising, in the direction from the N- to C-terminal end of theantibody, at least a hinge region, a CH2 region and a CH3 region. An Fcregion of the antibody may mediate the binding of the immunoglobulin tohost tissues or factors, including various cells of the immune system(such as effector cells) and components of the complement system.

The term “parent polypeptide” or “parent antibody”, is to be understoodas a polypeptide or antibody, which is identical to a polypeptide orantibody according to the invention, but where the parent polypeptide orparent antibody is without mutations, unless otherwise stated or clearlycontradicted by the context. For example, the antibody IgG1-CD27-A ofthe invention is the parent antibody of IgG1-CD27-A-P329R-E345R.

The term “hinge region” as used herein refers to the hinge region of animmunoglobulin heavy chain. Thus, for example the hinge region of ahuman IgG1 antibody corresponds to amino acids 216-230 according to theEu numbering (Eu-index) as set forth in Kabat, E.A. et al., Sequences ofproteins of immunological interest. 5th Edition - US Department ofHealth and Human Services, NIH publication No. 91-3242, pp 662,680,689(1991). However, the hinge region may also be any of the other subtypesas described herein.

The term “CH1 region” or “CH1 domain” as used herein refers to the CH1region of an immunoglobulin heavy chain. Thus, for example the CH1region of a human IgG1 antibody corresponds to amino acids 118-215according to the Eu numbering as set forth in Kabat (ibid). However, theCH1 region may also be any of the other subtypes as described herein.

The term “CH2 region” or “CH2 domain” as used herein refers to the CH2region of an immunoglobulin heavy chain. Thus, for example the CH2region of a human IgG1 antibody corresponds to amino acids 231-340according to the Eu numbering as set forth in Kabat (ibid). However, theCH2 region may also be any of the other subtypes as described herein.

The term “CH3 region” or “CH3 domain” as used herein refers to the CH3region of an immunoglobulin heavy chain. Thus, for example the CH3region of a human IgG1 antibody corresponds to amino acids 341-447according to the Eu numbering as set forth in Kabat (ibid). However, theCH3 region may also be any of the other subtypes as described herein.

The term “Fc-mediated effector functions” or “Fc effector functions” asused herein are used interchangeably and is intended to refer tofunctions that are a consequence of binding a polypeptide or antibody toits target or antigen on a cell membrane wherein the Fc-mediatedeffector function is attributable to the Fc region of the polypeptide orantibody. Examples of Fc-mediated effector functions include (i) C1qbinding, (ii) complement activation, (iii) complement-dependentcytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxity(ADCC), (v) Fc-gamma receptor (FcYR)-binding, (vi) antibody-dependent,FcyR-mediated antigen crosslinking, (vii) antibody-dependent cellularphagocytosis (ADCP), (viii) complement-dependent cellular cytotoxicity(CDCC), (ix) complement-enhanced cytotoxicity, (x) binding to complementreceptor of an opsonized antibody mediated by the antibody, (xi)opsonisation, and (xii) a combination of any of (i) to (xi).

The term “decreased Fc effector function(s)” or “Decreased Fc-mediatedeffector functions”, as used herein are used interchangeably and isintended to refer to an Fc effector function that is decreased for anantibody when directly compared to the Fc effector function of theparent polypeptide or antibody in the same assay.

The term “inertness”, “inert” or “non-activating” as used herein, refersto an Fc region which is at least not able to bind any FcyR, induceFc-mediated cross-linking of FcyRs, or induce FcyR-mediatedcross-linking of target antigens via two Fc regions of individualantibodies, or is not able to bind C1q. Thus, in certain embodiments ofthe invention the Fc region is inert. Therefore, in certain embodimentssome or all of the Fc-mediated effector functions are attenuated orcompletely absent.

The term “oligomerization”, as used herein, is intended to refer to aprocess that converts monomers to a finite degree of polymerization.Antibodies according to the invention can form oligomers, such ashexamers, via non-covalent association of Fc-regions after targetbinding, e.g., at a cell surface. Oligomerization of anti-CD27antibodies upon cell surface binding through Fc:Fc interactions mayincrease CD27 clustering resulting in activation of CD27 intracellularsignaling. The capacity of antibodies comprising the E345R or E430Gmutation to form oligomers, such as hexamers, upon cell surface bindingcan be evaluated as described in: de Jong RN et al, PLoS Biol. 2016 Jan6;14(1):e1002344. Fc-Fc-mediated oligomerization of antibodies occursafter target binding on a (cell) surface through the intermolecularassociation of Fc-regions between neighboring antibodies and isincreased by introduction of a E345R or a E430G mutation (numberingaccording to Eu-index).

The term “clustering”, as used herein, refers to oligomerization ofantibodies through non-covalent interactions.

The term “Fc-Fc enhancing”, as used herein, is intended to refer toincreasing the binding strength between, or stabilizing the interactionbetween, the Fc regions of two Fc-region containing antibodies so thatthe antibodies form oligomers such as hexamers on the cell surface. Thisenhancement can be obtained by certain amino acid mutations in the Fcregions of the antibodies, such as E345R or E430G. The term “monovalentantibody”, in the context of the present invention, refers to anantibody molecule that can interact with a specific epitope on anantigen, with only one antigen binding domain (e.g. one Fab arm). In thecontext of a bispecific antibody, “monovalent antibody binding” refersto the binding of the bispecific antibody to one specific epitope on anantigen with only one antigen binding domain (e.g. one Fab arm).

The term “monospecific antibody” in the context of the presentinvention, refers to an antibody that has binding specificity to oneepitope only. The antibody may be a monospecific, monovalent antibody(i.e. carrying only one antigen binding region) or a monospecifc,bivalent antibody (i.e. an antibody with two identical antigen bindingregions).

The term “bispecific antibody” refers to an antibody comprising twonon-identical antigen binding domains, e.g. two non-identical Fab-armsor two Fab-arms with non-identical CDR regions. In the context of thisinvention, bispecific antibodies have specificity for at least twodifferent epitopes. Such epitopes may be on the same or differentantigens or targets. If the epitopes are on different antigens, suchantigens may be on the same cell or different cells, cell types orstructures, such as extracellular matrix or vesicles and solubleprotein. A bispecific antibody may thus be capable of crosslinkingmultiple antigens, e.g. two different cells. A particular bispecificantibody of the present invention is capable of binding to CD27 and asecond target.

The term “bivalent antibody” refers to an antibody that has two antigenbinding regions, which bind to epitopes on one or two targets orantigens or binds to one or two epitopes on the same antigen. Hence, abivalent antibody may be a monospecific, bivalent antibody or abispecific, bivalent antibody.

The term “amino acid” and “amino acid residue” may herein be usedinterchangeably and are not to be understood limiting. Amino acids areorganic compounds containing amine (-NH₂) and carboxyl (-COOH)functional groups, along with a side chain (R group) specific to eachamino acid. In the context of the present invention, amino acids may beclassified based on structure and chemical characteristics. Thus,classes of amino acids may be reflected in one or both of the followingtables:

TABLE 1 Main classification based on structure and general chemicalcharacterization of R group Class Amino acid Acidic Residues D and EBasic Residues K, R, and H Hydrophilic Uncharged Residues S, T, N, and QAliphatic Uncharged Residues G, A, V, L, and I Non-polar UnchargedResidues C, M, and P Aromatic Residues F, Y, and W

TABLE 2 Alternative Physical and Functional Classifications of AminoAcid Residues Class Amino acid Hydroxyl group containing residues S andT Aliphatic residues I, L, V, and M Cycloalkenyl-associated residues F,H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, andY Negatively charged residues D and E Polar residues C, D, E, H, K, N,Q, R, S, and T Positively charged residues H, K, and R Small residues A,C, D, G, N, P, S, T, and V Very small residues A, G, and S Residuesinvolved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and TFlexible residues Q, T, K, S, G, P, D, E, and R

Substitution of one amino acid for another may be classified as aconservative or non-conservative substitution. In the context of theinvention, a “conservative substitution” is a substitution of one aminoacid with another amino acid having similar structural and/or chemicalcharacteristics, such substitution of one amino acid residue for anotheramino acid residue of the same class as defined in any of the two tablesabove: for example, leucine may be substituted with isoleucine as theyare both aliphatic, branched hydrophobes. Similarly, aspartic acid maybe substituted with glutamic acid since they are both small, negativelycharged residues.

In the context of the present invention, a substitution in an antibodyis indicated as:

Original amino acid – position – substituted amino acid;

Referring to the well-recognized nomenclature for amino acids, thethree-letter code, or one letter code, is used, including the codes“Xaa” or “X” to indicate any amino acid residue. Thus, Xaa or X maytypically represent any of the 20 naturally occurring amino acids. Theterm “naturally occurring” as used herein refers to any one of thefollowing amino acid residues; glycine, alanine, valine, leucine,isoleucine, serine, threonine, lysine, arginine, histidine, asparticacid, asparagine, glutamic acid, glutamine, proline, tryptophan,phenylalanine, tyrosine, methionine, and cysteine. Accordingly, thenotation “K409R” or “Lys409Arg” means, that the antibody comprises asubstitution of Lysine with Arginine in amino acid position 409.

Substitution of an amino acid at a given position to any other aminoacid is referred to as:

Original amino acid - position; or e.g. “K409”

For a modification where the original amino acid(s) and/or substitutedamino acid(s) may comprise more than one, but not all amino acid(s), themore than one amino acid may be separated by “,” or “/”. E.g. thesubstitution of Lysine with Arginine, Alanine, or Phenylalanine inposition 409 is:

“Lys409Arg,Ala,Phe” or “Lys409Arg/Ala/Phe” or “K409R,A,F” or “K409R/A/F”or “K409 to R, A, or F”.

Such designation may be used interchangeably in the context of theinvention but have the same meaning and purpose.

Furthermore, the term “a substitution” embraces a substitution into anyone or the other nineteen natural amino acids, or into other aminoacids, such as non-natural amino acids. For example, a substitution ofamino acid K in position 409 includes each of the followingsubstitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L,409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. This is,by the way, equivalent to the designation 409X, wherein the X designatesany amino acid other than the original amino acid. These substitutionsmay also be designated K409A, K409C, etc. or K409A,C, etc. orK409A/C/etc. The same applies by analogy to each and every positionmentioned herein, to specifically include herein any one of suchsubstitutions.

The antibody according to the invention may also comprise a deletion ofan amino acid residue. Such deletion may be denoted “del”, and includes,e.g., writing as K409del. Thus, in such embodiments, the Lysine inposition 409 has been deleted from the amino acid sequence.

The term “host cell”, as used herein, is intended to refer to a cellinto which an expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell, but also to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. Recombinant host cells include, forexample, transfectomas, such as CHO cells, HEK-293 cells, Expi293Fcells, PER.C6 cells, NS0 cells, and lymphocytic cells, and prokaryoticcells such as E. coli and other eukaryotic hosts such as plant cells andfungi.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing the antibody or a target antigen, such as CHOcells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plantcells, or fungi, including yeast cells.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gapsin Alignment).

The retention of similar residues may also or alternatively be measuredby a similarity score, as determined by use of a BLAST program (e.g.,BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62,Open Gap=11 and Extended Gap=1). Suitable variants typically exhibit atleast about 45%, such as at least about 55%, at least about 65%, atleast about 75%, at least about 85%, at least about 90%, at least about95%, or more (e.g., about 99%) similarity to the parent sequence.

The term “internalized” or “internalization” as used herein, refers to abiological process in which molecules such as the antibody according tothe present invention, are engulfed by the cell membrane and drawn intothe interior of the cell. Internalization may also be referred to as“endocytosis”.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, forinstance lymphocytes (such as B cells and T cells including cytolytic Tcells (CTLs)), killer cells, natural killer cells, macrophages,monocytes, eosinophils, polymorphonuclear cells, such as neutrophils,granulocytes, mast cells, and basophils. Some effector cells express Fcreceptors (FcgRs) or complement receptors and carry out specific immunefunctions. In some embodiments, an effector cell such as, e.g., anatural killer cell, is capable of inducing ADCC. For example,monocytes, macrophages, neutrophils, dendritic cells and Kupffer cellswhich express FcgRs, are involved in specific killing of target cellsand/or presenting antigens to other components of the immune system, orbinding to cells that present antigens. In some embodiments the ADCC canbe further enhanced by antibody driven classical complement activationresulting in the deposition of activated C3 fragments on the targetcell. C3 cleavage products are ligands for complement receptors (CRs),such as CR3, expressed on myeloid cells. The recognition of complementfragments by CRs on effector cells may promote enhanced Fcreceptor-mediated ADCC. In some embodiments antibody driven classicalcomplement activation leads to C3 fragments on the target cell. These C3cleavage products may promote direct complement-dependent cellularcytotoxicity (CDCC). In some embodiments, an effector cell mayphagocytose a target antigen, target particle or target cell which maydepend on antibody binding and mediated by FcyRs expressed by theeffector cells. The expression of a particular FcR or complementreceptor on an effector cell may be regulated by humoral factors such ascytokines. For example, expression of FcyRI has been found to beup-regulated by interferon y (IFN y) and/or G-CSF. This enhancedexpression increases the cytotoxic activity of FcyRI-bearing cellsagainst targets. An effector cell can phagocytose a target antigen orphagocytose or lyse a target cell. In some embodiments antibody drivenclassical complement activation leads to C3 fragments on the targetcell. These C3 cleavage products may promote direct phagocytosis byeffector cells or indirectly by enhancing antibody mediatedphagocytosis. In certain embodiments herein where the antibody has aninert Fc region the antibody does not induce an Fc-mediated effectorfunction.

“Effector T cells” or “Teffs” or “Teff” as used herein refers to Tlymphocytes that carry out a function of an immune response, such askilling tumor cells and/or activating an antitumor immune-response whichcan result in clearance of the tumor cells from the body. Examples ofTeff phenotypes include CD3⁺CD4⁺ and CD3⁺CD8⁺. Teffs may secrete,contain, or express markers such as IFNy, granzyme B and ICOS. It isappreciated that Teffs may not be fully restricted to these phenotypes.

“Memory T cells” as used herein refers to T lymphocytes that remain inthe body for a long period of time after an infection is removed.Examples of memory T cells include central memory T cells (CD45RA-CCR7+)and effector memory T cells (CD45RA-CCR7-). It is appreciated thatmemory T cells may not be fully restricted to these phenotypes.

“Regulatory T cells” or “Tregs” or “Treg” as used herein refers to Tlymphocytes that regulate the activity of other T cell(s) and/or otherimmune cells, usually by suppressing their activity. An example of aTreg phenotype is CD3⁺CD4⁺CD25⁺CD127dim. Tregs may further expressFoxp3. It is appreciated that Tregs may not be fully restricted to thisphenotype.

As used herein, the term “complement activation” refers to theactivation of the classical complement pathway, which is initiated by alarge macromolecular complex called C1 binding to antibody-antigencomplexes on a surface. C1 is a complex, which consists of 6 recognitionproteins C1q and a hetero-tetramer of serine proteases, C1r2C1s2. C1 isthe first protein complex in the early events of the classicalcomplement cascade that involves a series of cleavage reactions thatstarts with the cleavage of C4 into C4a and C4b and C2 into C2a and C2b.C4b is deposited and forms together with C2a an enzymatic activeconvertase called C3 convertase, which cleaves complement component C3into C3b and C3a, which forms a C5 convertase This C5 convertase splitsC5 in C5a and C5b and the last component is deposited on the membraneand that in turn triggers the late events of complement activation inwhich terminal complement components C5b, C6, C7, C8 and C9 assembleinto the membrane attack complex (MAC). The complement cascade resultsin the creation of pores in the cell membrane which causes lysis of thecell, also known as complement-dependent cytotoxicity (CDC). In certainembodiments herein where the antibody has an inert Fc region theantibody does not induce complement activation.

Complement activation can be evaluated by using C1q binding efficacy,CDC kinetics CDC assays (as described in WO2013/004842, WO2014/108198)or by the method Cellular deposition of C3b and C4b described inBeurskens et al., J Immunol Apr. 1, 2012 vol. 188 no. 7, 3532-3541.

The term “C1q binding” as used herein, is intended to refer to thebinding of C1q in the context of the binding of C1q to an antibody boundto its antigen. The antibody bound to its antigen is to be understood ashappening both in vivo and in vitro in the context described herein. C1qbinding can be evaluated for example by using antibody immobilized onartificial surfaces or by using antibody bound to a predeterminedantigen on a cellular or virion surface, as described in Example 8herein. The binding of C1q to an antibody oligomer is to be understoodherein as a multivalent interaction resulting in high avidity binding. Adecrease in C1q binding, for example resulting from the introduction ofa mutation in the antibody of the invention, may be measured bycomparing the C1q binding of the mutated antibody to the C1q binding ofits parent antibody (the antibody of the invention without the mutationwithin the same assay).

The term “treatment” refers to the administration of an effective amountof a therapeutically active antibody of the present invention with thepurpose of easing, ameliorating, arresting, or eradicating (curing)symptoms or disease states.

The term “effective amount” or “therapeutically effective amount” refersto an amount effective, at dosages and for periods of time necessary, toachieve a desired therapeutic result. A therapeutically effective amountof an antibody may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the antibodyto elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the antibody variant are outweighed by the therapeutically beneficialeffects.

The term “pharmacokinetic profile” as used herein can be determined asthe plasma IgG levels over time as described in Example 12 herein.

SPECIFIC EMBODIMENTS OF THE INVENTION

In a first aspect the invention provides an antibody comprising at leastone antigen-binding region capable of binding to human CD27 wherein saidantibody comprises a heavy chain variable (VH) region CDR1, CDR2, andCDR3 comprising the sequences as set forth in SEQ ID NOs: 5, 6, and 7,respectively, and a light chain variable (VL) region CDR1, CDR2, andCDR3 comprising the sequences as set forth in SEQ ID NO: 9, 10 and 11,respectively. In a further aspect the invention provides an antibodycomprising two of said antigen-binding regions comprising the VH regionCDR1, CDR2, and CDR3 comprising the sequences as set forth in SEQ IDNOs: 5, 6, and 7, respectively, and the VL region CDR1, CDR2, and CDR3comprising the sequences as set forth in SEQ ID NO: 9, 10 and 11respectively. Hereby anti-CD27 antibodies are provided which are able tobind to human CD27 and further to bind to a variant of human CD27comprising a mutation of A59T. In an embodiment of the invention theantibody binds CD27 e.g. on T cells and is agonistic upon binding to itstarget. Hereby an antibody is provided which stimulates the activationand proliferation of T-cells. The antibody may further stimulate memoryformation and survival of T-cells. Such an antibody is useful e.g. inthe treatment of cancer. The antibody is further capable of binding tocynomolgus CD27 which is useful for toxicological studies of theantibody.

It is well known in the art that mutations in the VH and VL of anantibody can be made to, for example, increase the affinity of anantibody to its target antigen, reduce its potential immunogenicityand/or to increase the yield of antibodies expressed by a host cell.Accordingly, in some embodiments, antibodies comprising variants of theCDR, VH and/or VL sequences of an antibody according to the inventionare also contemplated, particularly functional variants of the VH and/orVL region as set forth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.Functional variants may differ in one or more amino acids as compared tothe parent VH and/or VL sequence, e.g., in one or more CDRs, but stillallows the antigen-binding region to retain at least a substantialproportion (at least about 50 percent, 60 percent, 70 percent, 80percent, 90 percent, 95 percent or more) or even retain all of theaffinity and/or specificity of the parent antibody. Typically, suchfunctional variants retain significant sequence identity to the parentsequence. Exemplary variants include those which differ from therespective parent VH or VL region by 12 or less, such as 11, 10, 9, 8,7, 6, 5, 4, 3, 2 or 1 mutation(s) such as substitutions, insertions ordeletions of amino acid residues. Exemplary variants include those whichdiffer from the VH and/or VL and/or CDR regions of the parent sequencesmainly by conservative amino acid substitutions; for instance, 12, suchas 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the amino acid substitutionsin the variant can be conservative. In a further aspect of the inventionthe antibody may comprise at most 1, 2 or 3 mutations in the VH CDRregion and/or in the VL CDR region, respectively. Such mutations may besubstitutions. It is preferred that such substitutions do notsignificantly change the binding affinity and/or binding specificity ofthe anti-CD27 antibody of the invention. Accordingly, the presentinvention encompasses variants of the anti-CD27 antibody of theinvention which variants have the same functional features as theantibody comprising the VH region CDR sequences as set forth in SEQ IDNOs: 5, 6, and 7, and the VL region CDR sequences as set forth in SEQ IDNO: 9, 10 and 11.

In another aspect of the invention the antibody comprises a VH regioncomprising a sequence which is at least 80% identical to the VH regionas set forth in SEQ ID NO: 4. In another aspect of the invention theantibody comprises a VH region comprising a sequence which is at least85% identical to the VH region as set forth in SEQ ID NO: 4. In anotheraspect of the invention the antibody comprises a VH region comprising asequence which is at least 90% identical to the VH region as set forthin SEQ ID NO: 4. In another aspect of the invention the antibodycomprises a VH region comprising a sequence which is at least 95%identical to the VH region as set forth in SEQ ID NO: 4. In anotheraspect of the invention the antibody comprises a VH region comprising asequence which is at least 96% identical to the VH region as set forthin SEQ ID NO: 4. In another aspect of the invention the antibodycomprises a VH region comprising a sequence which is at least 97%identical to the VH region as set forth in SEQ ID NO: 4. In anotheraspect of the invention the antibody comprises a VH region comprising asequence which is at least 98% identical to the VH region as set forthin SEQ ID NO: 4. In another aspect of the invention the antibodycomprises a VH region comprising a sequence which is at least 99%identical to the VH region as set forth in SEQ ID NO: 4. In anotheraspect of the invention the antibody comprises a VH region comprising asequence as set forth in SEQ ID NO: 4.

In another aspect of the invention the antibody comprises a VH regioncomprising a sequence which is at least 80% identical to the VH regionas set forth in SEQ ID NO: 8. In another aspect of the invention theantibody comprises a VH region comprising a sequence which is at least85% identical to the VH region as set forth in SEQ ID NO: 8. In anotheraspect of the invention the antibody comprises a VH region comprising asequence which is at least 90% identical to the VH region as set forthin SEQ ID NO: 8. In another aspect of the invention the antibodycomprises a VH region comprising a sequence which is at least 95%identical to the VH region as set forth in SEQ ID NO: 8. In anotheraspect of the invention the antibody comprises a VH region comprising asequence which is at least 96% identical to the VH region as set forthin SEQ ID NO: 8. In another aspect of the invention the antibodycomprises a VH region comprising a sequence which is at least 97%identical to the VH region as set forth in SEQ ID NO: 8. In anotheraspect of the invention the antibody comprises a VH region comprising asequence which is at least 98% identical to the VH region as set forthin SEQ ID NO: 8. In another aspect of the invention the antibodycomprises a VH region comprising a sequence which is at least 99%identical to the VH region as set forth in SEQ ID NO: 8. In anotheraspect of the invention the antibody comprises a VH region comprising asequence as set forth in SEQ ID NO: 8.

In another aspect of the invention the antibody comprises the VH and VLregions comprising the sequences as set forth in SEQ ID NO: 4 and SEQ IDNO: 8, respectively.

In one aspect the antibody of the invention is an isolated antibody.

In one embodiment the antibody is a human antibody. In anotherembodiment the antibody is a humanized antibody. In another aspect theantibody is a chimeric antibody.

The antibody of the invention is in a preferred embodiment a full-lengthantibody. Accordingly, the antibody of the invention may furthercomprise a light chain constant region (CL) and a heavy chain constantregion (CH). The CH preferably comprises a CH1 region, a hinge region, aCH2 region and a CH3 region.

The antibody according to the invention may comprise a light chainconstant region which is a human kappa light chain. In another aspect itmay comprise a human lambda light chain constant region.

The antibody according to the invention may preferably further comprisea heavy chain constant region, which is of a human IgG isotype. It mayoptionally comprise a modified human IgG constant region. Such human IgGcomprise the Fc region which comprise the CH2 and CH3 region. Bymodifying the IgG constant region in the Fc region, it is for examplepossible to regulate the Fc effector functions of the antibody or toincrease the Fc-Fc interactions and thereby the antibodies tendency toform clusters such as hexamers. In one aspect of the invention the humanIgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4. Inone embodiment it is IgG1. In another aspect it is IgG2. In yet anotheraspect it is IgG3. In a further aspect it is IgG4. In one particularaspect the IgG is a modified human IgG comprising one or more amino acidsubstitutions in the Fc region. In one embodiment it may be a human IgG1comprising one or more amino acid substitutions in the Fc region. In afurther aspect of the invention the IgG1 comprises two or more aminoacid substitutions in the Fc region. In one embodiment the IgG1 Fcregion has two amino acid substitutions.

In a further aspect of the invention, the modified human IgG heavy chainconstant region comprises in the Fc region at most 10 amino acidsubstitutions. In another aspect it comprises at most 9 amino acidsubstitutions. In another aspect it comprises at most 8 amino acidsubstitutions. In another aspect it comprises at most 7 amino acidsubstitutions. In another aspect it comprises at most 6 amino acidsubstitutions. In another aspect it comprises at most 5 amino acidsubstitutions. In another aspect it comprises at most 4 amino acidsubstitutions. In another aspect it comprises at most 3 amino acidsubstitutions. In another aspect it comprises at most 2 amino acidsubstitutions in the Fc region.

Mutations in amino acid residues at positions corresponding to E430,E345 and S440 in a human IgG1 heavy chain, wherein the amino acidresidues are numbered according to the EU index, can improve the abilityof an antibody to induce CDC. Without being bound by theory, it isbelieved that by substituting one or more amino acid(s) in thesepositions, oligomerization of the antibody can be stimulated, therebymodulating Fc-mediated effector functions so as to, e.g., increase C1qbinding, complement activation, CDC, ADCP, internalization or otherrelevant function(s) that may provide in vivo efficacy.

The present invention in one aspect relates to a variant antibodycomprising an antigen-binding region and a variant Fc region.

In certain embodiments, an antibody variant binding to human CD27comprises:

-   (a) a heavy chain comprising a VH region comprising a VH CDR1    comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2    comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3    comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1    CH region comprising a mutation in one or more of E430, E345 and    S440, the amino acid residues being numbered according to the EU    index;-   (b) a light chain comprising a VL region comprising a VL CDR1    comprising the sequence as set forth in SEQ ID NO:9, a VL CDR2    comprising the sequence as set forth in SEQ ID NO:10, and a VL CDR3    comprising the sequence as set forth in SEQ ID NO:11.

In other certain embodiments, an antibody variant binding to human CD27comprises:

-   (a) a heavy chain comprising a VH region comprising SEQ ID NO:4 and    a human IgG1 CH region comprising a mutation in one or more of E430,    E345 and S440, the amino acid residues being numbered according to    the EU index, and-   (b) a light chain comprising a VL region comprising SEQ ID NO:8.

A variant antibody of the present invention comprises a variant Fcregion or a variant human IgG1 CH region comprising a mutation in one ormore of P329, E430 and E345. In the following, reference to themutations in the Fc region may similarly apply to the mutation(s) in thehuman IgG1 CH region and vice versa.

As described herein, the position of an amino acid to be mutated in theFc region can be given in relation to (i.e., “corresponding to”) itsposition in a naturally occurring (wildtype) human IgG1 heavy chain,when numbered according to the Eu index. So, if the parent Fc regionalready contains one or more mutations and/or if the parent Fc regionis, for example, an IgG2, IgG3 or IgG4 Fc region, the position of theamino acid corresponding to an amino acid residue such as, e.g., E430 ina human IgG1 heavy chain numbered according to the Eu index can bedetermined by alignment. Specifically, the parent Fc region is alignedwith a wild-type human IgG1 heavy chain sequence so as to identify theresidue in the position corresponding to E430 in the human IgG1 heavychain sequence. Any wildtype human IgG1 constant region amino acidsequence can be useful for this purpose, including any one of thedifferent human IgG1 allotypes set forth in Table 3.

In one aspect of the invention the modification in the IgG Fc regioninduces increased CD27 agonism compared to the identical antibody butcomprising a wild type IgG Fc region of the same isotype, such as IgG1.This may for example be obtained by introducing an amino acid other thanE at the amino acid position corresponding to position E345 and/or E430in a human IgG1 heavy chain according to Eu numbering. In one embodimentof the invention the amino acid residue at the position corresponding toposition E345 in a human IgG1 heavy chain according to Eu numbering isselected from the group comprising: A, C, D, F, G, H, I, K, L, M, N, Q,P, R, S, T, V, W and Y. In another aspect of the invention the aminoacid residue at the position corresponding to position E430 in a humanIgG1 heavy chain according to Eu numbering is selected from the groupcomprising: A, C, D, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W.

In a preferred embodiment the amino acid residue at the positioncorresponding to position E345 in a human IgG1 heavy chain according toEu numbering is R. Accordingly, the antibody of the invention maycomprise an E345R substitution in the Fc region. In another aspect ofthe invention the amino acid residue at the position corresponding toposition E430 in a human IgG1 heavy chain according to Eu numbering isG. Accordingly, the antibody of the invention may comprise a E430Gsubstitution in the Fc region. In another embodiment, the antibodycomprises an amino acid substitution selected from the group comprisingE430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y.

Hereby, antibodies are provided which have enhanced Fc-Fc interactionwhich may lead to antibody-dependent clustering of CD27 on the cellsurface upon antibody binding, thereby increasing the agonism of theantibody of the invention.

In another embodiment of the antibody of the invention the amino acidresidue at the position corresponding to position P329 in a human IgG1heavy chain according to Eu numbering is substituted with an amino acidselected from the group comprising: A, C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W and Y. Accordingly, the antibody of the invention mayfurther comprise a mutation in position 329.

In a further aspect of the invention the antibody has the amino acidresidue R at the position corresponding to position P329 in a human IgG1heavy chain according to Eu numbering. Accordingly, the antibody of theinvention may have a P329R substitution in the Fc region. Without beingbound by theory, it is believed that the antibody of the inventioncomprising an E345R mutation in the Fc region (as e.g. set out in SEQ IDNO: 13) has increased serum clearance. The inventors found that furtherintroducing a mutation at position 329, such as P329R (as e.g. set outin SEQ ID NO: 15) restored the clearance of the antibody of theinvention to the level of the antibody comprising a wt IgG1 as e.g. setout in SEQ ID NO: 12.

In another preferred embodiment the amino acid residues at the positionscorresponding to positions P329 and E345 in a human IgG1 heavy chainaccording to Eu numbering are both R. Hereby an antibody is providedwhich has increased CD27 receptor agonism and comparable pharmacokineticproperties, such as e.g. serum clearance, when compared to an antibodycomprising the same VH and VL region and comprising an identical IgG1heavy chain constant region with the exception of comprising thewildtype amino acid P at position 329 and the wildtype amino acid E atposition 345.

Thus, in an embodiment the invention provides a CD27 binding antibodywhich has increased receptor agonism upon binding to CD27 and whichfurther has pharmacokinetic properties which are comparable, such assimilar or even identical pharmacokinetic properties, when compared tothe pharmacokinetic properties of an antibody comprising the same VH andVL region but comprising a wild type IgG1 heavy chain constant regionsuch as e.g. set out in SEQ ID NO: 12. In other words the inventionprovides a CD27 binding antibody which has pharmacokinetic propertieswhich are not significantly different than the pharmacokineticproperties of an identical CD27 binding antibody except for comprising awild type IgG1 heavy chain constant region.

In other embodiments of the invention the antibody comprises a variantFc region according to any one of the preceding sections, which variantFc region is a variant of a human IgG Fc region selected from the groupconsisting of a human IgG1, IgG2, IgG3 and IgG4 Fc region. That is, themutation in one or more of the amino acid residues corresponding to E430and E345 and P329 is/are made in a parent Fc region which is a human IgGFc region selected from the group consisting of an IgG1, IgG2, IgG3 andIgG4 Fc region. Preferably, the parent Fc region is a naturallyoccurring (wildtype) human IgG Fc region, such as a human wildtype IgG1,IgG2, IgG3 or IgG4 Fc region, or a mixed isotype thereof. Thus, thevariant Fc region may, except for the recited mutation (in one or moreof the amino acid residues selected from E430 and E345 and P329), be ahuman IgG1, IgG2, IgG3 or IgG4 isotype, or a mixed isotype thereof.

In one embodiment, the parent Fc region and/or human IgG1 CH region is awild-type human IgG1 isotype.

Thus, the variant Fc region may except for the recited mutation (in E430or E345 or P329), be a human IgG1 Fc region.

In a specific embodiment, the parent Fc region and/or human IgG1 CHregion is a human wild-type IgG1m(f) isotype.

In a specific embodiment, the parent Fc region and/or human IgG1 CHregion is a human wild-type IgG1m(z) isotype.

In a specific embodiment, the parent Fc region and/or human IgG1 CHregion is a human wild-type IgG1m(a) isotype.

In a specific embodiment, the parent Fc region and/or human IgG1 CHregion is a human wild-type IgG1m(x) isotype.

In a specific embodiment, the parent Fc region and/or human IgG1 CHregion is a human wild-type IgG1 of a mixed allotype, such as IgG1m(za),IgG1m(zax), IgG1m(fa), or the like.

Thus, the variant Fc region and/or human IgG1 CH region may, except forthe recited mutation (in E430 or E345 or P329), be a human IgG1m(f),IgG1m(a), IgG1m(x), IgG1m(z) allotype or a mixed allotype of any two ormore thereof.

In a specific embodiment, the parent Fc region and/or human IgG1 CHregion is a human wild-type IgG1m(za) isotype.

In a specific embodiment, the parent Fc region is a human wild-type IgG2isotype.

In a specific embodiment, the parent Fc region is a human wild-type IgG3isotype.

In a specific embodiment, the parent Fc region is a human wild-type IgG4isotype.

CH region amino acid sequences of specific examples of wild-type humanIgG isotypes and IgG1 allotypes are set forth in Table 3.

In another aspect the invention provides an antibody which comprises aheavy chain constant region comprising an amino acid sequence selectedfrom the group comprising: SEQ ID Nos 12, 13, 14, 15, 18, 19, 20, 21,22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and 36. In one aspect the heavychain constant region has the amino acid sequence of SEQ ID NO: 12. Inone aspect the heavy chain constant region has the amino acid sequenceof SEQ ID NO: 13. In one aspect the heavy chain constant region has theamino acid sequence of SEQ ID NO: 14. In one aspect the heavy chainconstant region has the amino acid sequence of SEQ ID NO: 15. In oneaspect the heavy chain constant region has the amino acid sequence ofSEQ ID NO: 18. In one aspect the heavy chain constant region has theamino acid sequence of SEQ ID NO: 19. In one aspect the heavy chainconstant region has the amino acid sequence of SEQ ID NO: 20. In oneaspect the heavy chain constant region has the amino acid sequence ofSEQ ID NO: 21. In one aspect the heavy chain constant region has theamino acid sequence of SEQ ID NO: 22. In one aspect the heavy chainconstant region has the amino acid sequence of SEQ ID NO: 23. In oneaspect the heavy chain constant region has the amino acid sequence ofSEQ ID NO: 27. In one aspect the heavy chain constant region has theamino acid sequence of SEQ ID NO: 28. In one aspect the heavy chainconstant region has the amino acid sequence of SEQ ID NO: 29. In oneaspect the heavy chain constant region has the amino acid sequence ofSEQ ID NO: 30. In one aspect the heavy chain constant region has theamino acid sequence of SEQ ID NO: 31. In one aspect the heavy chainconstant region has the amino acid sequence of SEQ ID NO: 32. In oneaspect the heavy chain constant region has the amino acid sequence ofSEQ ID NO: 33. In one aspect the heavy chain constant region has theamino acid sequence of SEQ ID NO: 34. In one aspect the heavy chainconstant region has the amino acid sequence of SEQ ID NO: 36.

In an embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 15 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 12 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 13 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 14 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 18 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 19 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 20 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 21 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 22 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 23 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 27 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 28 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 29 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 30 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 31 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 32 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 33 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 34 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In another embodiment the antibody according to the invention comprises:

-   a. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   b. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   c. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 36 and-   d. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 16.

In alternative embodiments of the above antibodies the CL region may bethe amino acid sequence set forth in SEQ ID No: 17.

In an embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 15 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 12 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 13 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 14 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 18 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 19 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 20 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 21 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 22 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 23 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 27 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 28 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 29 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 30 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 31 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 32 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 33 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 34 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprises:

-   e. The VH region comprising the amino acid sequence set forth in SEQ    ID No: 4-   f. The VL region comprising the amino acid sequence set forth in SEQ    ID No: 8-   g. The CH region comprising the amino acid sequence set forth in SEQ    ID No: 36 and-   h. The CL region comprising the amino acid sequence set forth in SEQ    ID No: 17.

In another embodiment the antibody according to the invention comprisesa heavy chain comprising the amino acid sequence set forth in SEQ ID NO:24 and a light chain comprising the amino acid sequence set forth in SEQID NO: 25.

In another embodiment the antibody according to the invention comprisesa heavy chain comprising the amino acid sequence set forth in SEQ ID NO:35 and a light chain comprising the amino acid sequence set forth in SEQID NO: 25.

In yet another aspect the invention provides an antibody which comprisesa heavy chain constant region that is modified so that the antibodyinduces an Fc-mediated effector function to a lesser extent relative toan identical antibody except for the modification. An example hereof isthe CD27 binding antibody of the invention comprising a P329R and anE345R substitution. Such antibody induces one or more Fc-mediatedeffector function(s) to a lesser extent compared to the antibodycomprising the same sequence except not comprising the P329Rsubstitution and also compared to the same antibody comprising the samesequence except not comprising the P329R and E345R substitutions, suchas a wildtype IgG1 heavy chain. In one embodiment the Fc-mediatedeffector function is decreased by at least 20%. In another aspect theFc-mediated effector function is decreased by at least 30%. In anotheraspect the Fc-mediated effector function is decreased by at least 40%.In another aspect the Fc-mediated effector function is decreased by atleast 50%. In another aspect the Fc-mediated effector function isdecreased by at least 60%. In another aspect the Fc-mediated effectorfunction is decreased by at least 70%. In another aspect the Fc-mediatedeffector function is decreased by at least 80%. In another aspect theFc-mediated effector function is decreased by at least 90%. In anotheraspect the antibody does not induce one or more Fc-mediated effectorfunctions. The one or more Fc-effector functions that are decreased ornot at all induced may be selected from the following group:complement-dependent cytotoxicity (CDC), complement-dependentcell-mediated cytotoxicity (CDCC), complement activation,antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependentcell-mediated phagocytosis (ADCP), C1q binding and FcyR binding.Accordingly, in one embodiment the antibody of the invention induces CDCto a degree which is decreased by at least 20%, such as at least 30%, orat least 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or decreased by at least 90% relative to the identicalantibody but a wildtype IgG1 HC constant region. In another embodimentthe antibody of the invention does not induce CDC.

In another aspect, the antibody of the invention induces CDCC to adegree which is decreased by at least 20%, such as at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or decreased by at least 90% relative to the identicalantibody but having a wildtype IgG1 HC constant region. In anotherembodiment the antibody of the invention does not induce CDCC.

In another aspect, the antibody of the invention induces ADCC to adegree which is decreased by at least 20%, such as at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or decreased by at least 90% relative to the identicalantibody but having a wildtype IgG1 HC constant region. In anotherembodiment the antibody of the invention does not induce ADCC.

In another aspect, the antibody of the invention induces ADCP to adegree which is decreased by at least 20%, such as at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or decreased by at least 90% relative to the identicalantibody but having a wildtype IgG1 HC constant region. In anotherembodiment the antibody of the invention does not induce ADCP.

In another aspect, the antibody of the invention induces C1q binding toa degree which is decreased by at least 20%, such as at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or decreased by at least 90% relative to the identicalantibody but having a wildtype IgG1 HC constant region. In anotherembodiment the antibody of the invention does not induce C1q binding.

Preferably the C1q binding is determined as in example 8.

In another aspect, the antibody of the invention induces FcyR binding toa degree which is decreased by at least 20%, such as at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or decreased by at least 90% relative to the identicalantibody but having a wildtype IgG1 HC constant region. In anotherembodiment the antibody of the invention does not induce FcyR binding.Preferably the FcyR binding is determined as in example 9.

In one embodiment the antibody of the invention has reduced C1q bindingand reduced FcyR binding compared to the antibody comprising the sameamino acid sequences except not comprising the P329R substitution.

In one embodiment, the antibody according to any aspect or embodimentherein is, except for the recited mutations, a human antibody.

In an embodiment of the invention the antibody is a monovalent antibody.

In another embodiment the antibody is a bivalent antibody.

Further, the antibody of the invention may be a monospecific antibody.

In one embodiment, the antibody according to any aspect or embodimentherein is a monoclonal antibody, such as a human monoclonal antibody,such as a human bivalent monoclonal antibody, such as a human bivalentfull-length monoclonal antibody.

In a preferred embodiment, the antibody according to any aspect orembodiment herein is, except for the optional recited mutations in theFc region, an IgG1 antibody, such as a full length IgG1 antibody, suchas a human full-length IgG1 antibody, optionally a human monoclonalfull-length bivalent IgG1,κ antibody, e.g. a human monoclonalfull-length bivalent IgG1m(f),κ antibody.

An antibody according to the present invention is advantageously in abivalent monospecific format, comprising two antigen-binding regionsbinding to the same epitope. However, bispecific formats where one ofthe antigen-binding regions binds to a different epitope are alsocontemplated. So, the antibody according to any aspect or embodimentherein can, unless contradicted by context, be either a monospecificantibody or a bispecific antibody.

Accordingly, in another embodiment, the antibody of the invention is abispecific antibody comprising a first antigen binding region capable ofbinding human CD27 as described herein and comprising a second antigenbinding region capable of binding to a different epitope on human CD27.In another embodiment, the antibody of the invention is a bispecificantibody comprising a first antigen binding region capable of bindinghuman CD27 as described herein and comprising a second antigen bindingregion capable of binding a different target. Such target may be on adifferent cell or on the same cell as CD27.

In an aspect of the invention the antibody is capable of binding tohuman CD27 having the sequence as set forth in SEQ ID NO: 1. However,human CD27 may in some individuals be expressed as a variant hereof.Thus, in another aspect the antibody of the invention is further capableof binding to a human CD27 variant, such as for example the human CD27variant as set forth in SEQ ID NO: 2. In another embodiment, theantibody of the invention if further capable of binding to cynomolgusCD27, such as set forth in SEQ ID NO: 3.

In a further embodiment of the invention the antibody is capable ofbinding CD27-expressing human T cells.

In another embodiment of the invention the antibody is capable ofbinding CD27-expressing cynomolgus T cells.

In one embodiment of the invention the full length IgG1 antibody has hadthe C-terminal Lysine of the HC cleaved off. Such an antibody is alsoconsidered a “full length antibody”.

In another embodiment of the invention the antibody is capable ofinducing proliferation of human T cells such as CD4⁺ and CD8⁺ T-cells,such as T helper cells and cytotoxic T cells. Such activity may beassayed as described in Example 6 or 7 herein.

In another embodiment of the invention the antibody is capable ofinducing activation of human CD27-expressing Jurkat reporter T cellssuch as described in Example 2 herein.

In another embodiment of the invention the antibody is capable ofinducing activation of human CD27-expressing Jurkat reporter T cells inthe absence of Fcy receptor IIb cross-linking such as described inExample 11 herein.

In another embodiment of the invention the antibody is capable ofinducing proliferation of CD4+ and CD8⁺ T cells with a central memory Tcell phenotype.

In another embodiment of the invention the antibody is capable ofinducing IFN gamma production.

Antibodies are well known as therapeutics which may be used in treatmentof various diseases. Another method for administration of an antibody toa subject in need thereof includes administration of a nucleic acid or acombination of nucleic acids encoding said antibody for in vivoexpression of said antibody.

Hence, in one aspect, the present invention also relates to a nucleicacid encoding the heavy chain of an antibody according to the presentinvention, wherein said heavy chain comprises a VH region comprising aVH CDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1 CHregion.

In another aspect, the present invention also relates to a nucleic acidencoding the heavy chain of an antibody according to the presentinvention, wherein said heavy chain comprises a VH region comprising aVH CDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1 CHregion with a mutation in one or both of E430 and E345, the amino acidresidues being numbered according to the Eu index.

In another aspect, the present invention also relates to a nucleic acidencoding the heavy chain of an antibody according to the presentinvention, wherein said heavy chain comprises a VH region comprising aVH CDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1 CHregion with a mutation in one or both of P329 and E345, the amino acidresidues being numbered according to the Eu index.

In one aspect the present invention also relates to a nucleic acid or acombination of nucleic acids, encoding an antibody according to thepresent invention.

In some embodiments the present invention relates to a nucleic acid or acombination of nucleic acids encoding an antibody comprising:

-   a) an antigen-binding region comprising a VH CDR1 comprising the    sequence as set forth in SEQ ID NO:5, a VH CDR2 comprising the    sequence as set forth in SEQ ID NO:6, a VH CDR3 comprising the    sequence as set forth in SEQ ID NO:7, a VL CDR1 comprising the    sequence as set forth in SEQ ID NO:9, a VL CDR2 comprising the    sequence as set forth in SEQ ID NO:10, and a VLCDR3 comprising the    sequence as set forth in SEQ ID NO:11, and-   b) a variant Fc region comprising a mutation in one or both amino    acids corresponding to P329 and E345 and in a human IgG1 heavy    chain, wherein the amino acid residues are numbered according to the    Eu index.

In one embodiment, the antibody of the present invention is encoded byone nucleic acid. Thus, the nucleotide sequences encoding the antibodyof the present invention are present in one nucleic acid sequence or thesame nucleic acid molecule.

In another embodiment the antibody of the present invention is encodedby a combination of nucleic acid sequences, typically by two nucleicacid sequences. In one embodiment said combination of nucleic acidsequences comprise a nucleic acid sequence encoding the heavy chain ofsaid antibody and a nucleic acid sequence encoding the light chain ofsaid antibody.

In some embodiments the present invention relates to a nucleic acidsequence or a combination of nucleic acid sequences encoding an antibodycomprising:

-   a) a heavy chain comprising a VH region comprising a VH CDR1    comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2    comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3    comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1    CH region comprising a mutation in one or both of P329 and E345, the    amino acid residues being numbered according to the Eu index;-   b) a light chain comprising a VL region comprising a VL CDR1    comprising the sequence as set forth in SEQ ID NO:9, a VL CDR2    comprising the sequence as set forth in SEQ ID NO:10, and a VL CDR3    comprising the sequence as set forth in SEQ ID NO:11.

In one embodiment, the antibody of the present invention is encoded byone nucleic acid. Thus, the nucleotide sequences encoding the antibodyof the present invention are present in one nucleic acid or the samenucleic acid molecule.

In another embodiment the antibody of the present invention is encodedby a combination of nucleic acid sequences, typically by two nucleicacid sequences. In one embodiment said combination of nucleic acidsequences comprise a nucleic acid sequence encoding the heavy chain ofsaid antibody and a nucleic acid sequence encoding the light chain ofsaid antibody.

As described above the nucleic acid sequences may be used as a mean forsupplying therapeutic proteins, such as antibodies, to a subject in needthereof.

In some embodiments, said nucleic acid may be deoxyribonucleic acid(DNA). DNAs and methods of preparing DNA suitable for in vivo expressionof therapeutic proteins, such as antibodies are well known to a personskilled in the art and include but is not limited to that described byPatel A et al., 2018, Cell Reports 25, 1982-1993.

In some embodiments, said nucleic acid may be ribonucleic acid (RNA),such as messenger RNA (mRNA). In some embodiments, the mRNA may compriseonly naturally occurring nucleotides. In some embodiments the mRNA maycomprise modified nucleotides, wherein modified refers to saidnucleotides being chemically different from the naturally occurringnucleotides. In some embodiments the mRNA may comprise both naturallyoccurring and modified nucleotides.

Different nucleic acid sequences suitable for in vivo expression oftherapeutic proteins, such as antibodies, in a subject are well known toa person skilled in the art. For example, a mRNA suitable for expressionof a therapeutic antibody in a subject, often comprise an Open ReadingFrame (ORF), flanked by Untranslated Regions (UTRs) comprising specificsequences, and 5′and 3′ends being formed by a cap structure and apoly(A)tail (see e.g. Schlake et al., 2019, Molecular Therapy Vol. 27 No4 April).

Examples of methods for optimization of RNA and RNA molecules suitable,e.g. mRNA, for in vivo expression include, but are not limited to thosedescribed in US9254311; US9221891; US20160185840 and EP3118224.

Naked nucleic acid sequence(s) which are administered to a subject forin vivo expression are prone to degradation and/or of causing animmunogenic response in the subject. Furthermore, for in vivo expressionof the antibody encoded by the nucleic acid sequences said nucleic acidsequences typically is administered in a form suitable for the nucleicacid sequences to enter the cells of the subject. Different methods fordelivering a nucleic acid sequence for in vivo expression exist andinclude both methods involving mechanical and chemical means. Forexample, such methods may involve electroporation or tattooing thenucleic acid onto the skin (Patel et al., 2018, Cell Reports 25,1982-1993). Other methods suitable for administration of the nucleicacid sequences to a subject involve administration of the nucleic acidin a suitable formulation. Thus, the present invention also relates to adelivery vehicle comprising a nucleic acid of the present invention.

In some embodiments, said delivery vehicle may comprise a nucleic acidsequence encoding a heavy chain of an antibody according to the presentinvention. Thus in one embodiment said nucleic acid sequence may encodea heavy chain comprising a VH region comprising a VH CDR1 comprising thesequence as set forth in SEQ ID NO:5, a VH CDR2 comprising the sequenceas set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence as setforth in SEQ ID NO:7 and a human IgG1 CH region with a mutation in oneor both of P329 and E345, the amino acid residues being numberedaccording to the Eu index.

In some embodiments, the present invention also relates to a deliveryvehicle comprising a nucleic acid sequence encoding a light chain of anantibody according to the present invention. Thus, in one embodimentsaid nucleic acid sequence may encode a light chain comprising a VLregion comprising a VL CDR1 comprising the sequence as set forth in SEQID NO:9, a VL CDR2 comprising the sequence as set forth in SEQ ID NO:10,and a VL CDR3 comprising the sequence as set forth in SEQ ID NO:11.

The present invention also relates to a mixture of delivery vehiclescomprising a delivery vehicle comprising a nucleic acid sequenceencoding a heavy chain of an antibody according to the present inventionand delivery vehicle comprising a nucleic acid sequence encoding a lightchain of an antibody according to the present invention. Thus in oneembodiment said mixture of delivery vehicles comprise a delivery vehiclecomprising a nucleic acid sequence encoding a heavy chain comprising aVH region comprising a VH CDR1 comprising the sequence as set forth inSEQ ID NO:5, a VH CDR2 comprising the sequence as set forth in SEQ IDNO:6, a VH CDR3 comprising the sequence as set forth in SEQ ID NO:7 anda human IgG1 CH region with a mutation in one or both of E430 and E345,the amino acid residues being numbered according to the Eu index; and adelivery vehicle comprising a nucleic acid sequence encoding a lightchain comprising a VL region comprising a VL CDR1 comprising thesequence as set forth in SEQ ID NO:9, a VL CDR2 comprising the sequenceas set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence asset forth in SEQ ID NO:11.

In some embodiments, said delivery vehicle comprises a nucleic acidsequence or a combination of nucleic acid sequences encoding the heavyand a nucleic light chain of an antibody according to the presentinvention.

Thus in one embodiment said delivery vehicle may comprise a nucleic acidsequence encoding a heavy chain comprising a VH region comprising a VHCDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1 CHregion with a mutation in one or both of E430 and E345 the amino acidresidues being numbered according to the Eu index; and a light chaincomprising a VL region comprising a VL CDR1 comprising the sequence asset forth in SEQ ID NO:9, a VL CDR2 comprising the sequence as set forthin SEQ ID NO:10, and a VL CDR3 comprising the sequence as set forth inSEQ ID NO:11.

In yet an embodiment said delivery vehicle may comprise a nucleic acidsequence encoding a heavy chain comprising a VH region comprising a VHCDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1 CHregion with the mutations P329R and E345R the amino acid residues beingnumbered according to the Eu index; and a light chain comprising a VLregion comprising a VL CDR1 comprising the sequence as set forth in SEQID NO:9, a VL CDR2 comprising the sequence as set forth in SEQ ID NO:10,and a VL CDR3 comprising the sequence as set forth in SEQ ID NO:11.

In another embodiment said delivery vehicle may comprise a nucleic acidsequence encoding a heavy chain comprising a VH region comprising a VHCDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a WT human IgG1CH region; and a light chain comprising a VL region comprising a VL CDR1comprising the sequence as set forth in SEQ ID NO:9, a VL CDR2comprising the sequence as set forth in SEQ ID NO:10, and a VL CDR3comprising the sequence as set forth in SEQ ID NO:11.

Thus, the nucleic acid sequences encoding the heavy and light chain ofthe antibody according to the present invention are present in one (thesame) nucleic acid molecule.

In another embodiment said delivery vehicle may comprise a nucleic acidsequence encoding a heavy chain comprising a VH region comprising a VHCDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a WT human IgG1CH region; and a nucleic acid encoding a light chain comprising a VLregion comprising a VL CDR1 comprising the sequence as set forth in SEQID NO:9, a VL CDR2 comprising the sequence as set forth in SEQ ID NO:10,and a VL CDR3 comprising the sequence as set forth in SEQ ID NO:11.

In another embodiment said delivery vehicle may comprise a nucleic acidsequence encoding a heavy chain comprising a VH region comprising a VHCDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1 CHregion with a mutation in one or both of E430 and E345 the amino acidresidues being numbered according to the Eu index; and a nucleic acidsequence encoding a light chain comprising a VL region comprising a VLCDR1 comprising the sequence as set forth in SEQ ID NO:9, a VL CDR2comprising the sequence as set forth in SEQ ID NO:10, and a VL CDR3comprising the sequence as set forth in SEQ ID NO:11.

In another embodiment said delivery vehicle may comprise a nucleic acidsequence encoding a heavy chain comprising a VH region comprising a VHCDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7 and a human IgG1 CHregion with the mutations of P329R and E345R the amino acid residuesbeing numbered according to the Eu index; and a nucleic acid sequenceencoding a light chain comprising a VL region comprising a VL CDR1comprising the sequence as set forth in SEQ ID NO:9, a VL CDR2comprising the sequence as set forth in SEQ ID NO:10, and a VL CDR3comprising the sequence as set forth in SEQ ID NO:11.

Thus, the nucleic acid sequences encoding the heavy and light chain ofthe antibody variant according to the present invention are present onseparate or different nucleic acid molecules.

In some embodiments said delivery vehicle may be a lipid formulation.The lipids of the formulation may particle(s), such as a lipidnanoparticle(s) (LNPs). The nucleic acid sequence or combination ofnucleic acid sequences of the present may be encapsulated within saidparticle, e.g. within said LNP.

Different lipid formulations suitable for administration of a nucleicacid to a subject for in vivo expression are well known to a personskilled in the art. For example, said lipid formulation may typicallycomprise lipids, ionizable amino lipids, PEG-lipids, cholesterol or anycombination thereof.

Various forms and methods for preparation of lipid formulations suitablefor administration of a nucleic acid sequence to a subject forexpression of a therapeutic antibody are well known in the art. Examplesof such lipid formulations include but are not limited to thosedescribed in US20180170866 (Arcturus), EP 2391343 (Arbutus), WO2018/006052 (Protiva), WO2014152774 (Shire Human Genetics), EP 2 972 360(Translate Bio), US10195156 (Moderna), and US20190022247 (Acuitas).

The invention also provides isolated nucleic acid sequences and vectorsencoding an antibody variant according to any one of the aspects andembodiments described herein, as well as vectors and expression systemsencoding the variants. Suitable nucleic acid constructs, vectors andexpression systems for antibodies and variants thereof are known in theart, and include, but are not limited to, those described in theExamples. In embodiments where the variant antibody comprises HC and LCthat are separate polypeptides rather than contained in a singlepolypeptide (e.g., as in a scFv-Fc fusion protein), the nucleotidesequences encoding the heavy and light chains may be present in the sameor different nucleic acids or vectors.

Accordingly, in one aspect the invention provides an isolated nucleicacid sequence or a combination of nucleic acid sequences encoding theantibody according to any aspect or embodiment herein. The inventionalso provides a nucleic acid sequence encoding a VH region comprising aVH CDR1 comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7.

Further, the invention provides a nucleic acid sequence encoding a VLregion comprising a VL CDR1 comprising the sequence as set forth in SEQID NO:9, a VL CDR2 comprising the sequence as set forth in SEQ ID NO:10,a VL CDR3 comprising the sequence as set forth in SEQ ID NO:11. Further,the invention provides a nucleic acid sequence encoding a VH regioncomprising the amino acid sequence as set forth in SEQ ID NO: 4. Theinvention also relates to a nucleic acid sequence encoding a VL regioncomprising the amino acid sequence as set forth in SEQ ID NO: 8.

In a further aspect the invention provides a nucleic acid sequenceencoding the heavy chain of the antibody according to any aspect orembodiment descried herein. In a further aspect the invention provides anucleic acid sequence encoding the light chain of the antibody accordingto any aspect or embodiment descried herein. In another aspect theinvention relates to a nucleic acid sequence encoding a heavy chaincomprising a VH region comprising the sequence as set forth in SEQ IDNO:4 and a human IgG1 CH region comprising a mutation of P329 and/or ofE345, with the amino acid residues being numbered according to the Euindex. In yet another aspect the invention provides a nucleic acidsequence encoding a light chain comprising a VL region comprising thesequence as set forth in SEQ ID NO:8 and a human kappa constant regioncomprising the sequence as set forth in SEQ ID NO:16. In yet anotheraspect the invention provides a nucleic acid sequence encoding a lightchain comprising a VL region comprising the sequence as set forth in SEQID NO:8 and a human lambda constant region comprising the sequence asset forth in SEQ ID NO:17.

In an embodiment of the invention the nucleic acid sequence orcombination of nucleic acid sequences are RNA or DNA. In an embodimentof the invention the nucleic acid sequence or combination of nucleicacid sequences is/are mRNA.

The invention further provides an expression vector comprising thenucleic acid sequence or combination thereof according to any aspect orembodiment described herein.

In another aspect the invention relates to a nucleic acid sequence or acombination of nucleic acid sequences as described herein for use inexpression in mammalian cells.

In a further embodiment the invention relates to a recombinant hostcell, which produces an antibody as defined herein, optionally whereinthe host cell comprises the expression vector described above. Incertain embodiments the recombinant host cell is a eukaryotic orprokaryotic cell.

In another aspect the invention relates to a method of producing anantibody according to any aspect or embodiment herein, comprisingcultivating the recombinant host cell as described above in a culturemedium and under conditions suitable for producing the antibody and,optionally, purifying or isolating the antibody from the culture medium.

In one aspect, the invention relates to a nucleic acid or an expressionvector comprising

-   (i) a nucleotide sequence encoding a heavy chain sequence of an    antibody according to any one of the embodiments disclosed herein;-   (ii) a nucleotide sequence encoding a light chain sequence of an    antibody according to any one of the embodiments disclosed herein;    or-   (iii) both (i) and (ii).

In one aspect, the invention relates to a nucleic acid or an expressionvector comprising a nucleotide sequence encoding a heavy chain sequenceof an antibody variant according to any one of the embodiments disclosedherein.

In one aspect, the invention relates to a nucleic acid sequence or anexpression vector comprising a nucleotide sequence encoding a heavychain sequence and a light chain sequence of an antibody according toany one of the embodiments disclosed herein.

In one aspect, the invention relates to a combination of a first and asecond nucleic acid or a combination of a first and second expressionvector, optionally in the same host cell, where the first comprises anucleotide sequence according to (i), and the second comprises anucleotide sequence according to (ii).

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, anucleic acid is comprised in a naked DNA or RNA vector, including, forexample, a linear expression element (as described in for instance Sykesand Johnston, Nat Biotech 17, 355 59 (1997)), a compacted nucleic acidvector (as described in for instance US 6,077, 835 and/or WO 00/70087),a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge”minimally-sized nucleic acid vector (as described in for instanceSchakowski et al., Mol Ther 3, 793 800 (2001)), or as a precipitatednucleic acid vector construct, such as a CaP04-precipitated construct(as described in for instance WO200046147, Benvenisty and Reshef, PNASUSA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraroand Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acidvectors and the usage thereof are well known in the art (see forinstance US 5,589,466 and US 5,973,972).

In one embodiment, the vector is suitable for expression of the antibodyin a bacterial cell. Examples of such vectors include expression vectorssuch as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, JBiol Chem 264, 5503 5509 (1989), pET vectors (Novagen, Madison Wl) andthe like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), and Grant et al., Methods in Enzymol 153,516 544 (1987)).

An expression vector may also or alternatively be a vector suitable forexpression in mammalian cells, e.g. a vector comprising glutaminesynthetase as a selectable marker, such as the vectors described inBebbington (1992) Biotechnology (NY) 10:169-175.

A nucleic acid and/or vector may also comprise a nucleic acid sequenceencoding a secretion/localization sequence, which can target apolypeptide, such as a nascent polypeptide chain, to the periplasmicspace or into cell culture media. Such sequences are known in the artand include secretion leader or signal peptides.

The expression vector may comprise or be associated with any suitablepromoter, enhancer, and other expression-facilitating elements. Examplesof such elements include strong expression promoters (e. g., human CMVIE promoter/enhancer as well as RSV, SV40, SL3 3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE.

In one embodiment, the antibody-encoding expression vector may bepositioned in and/or delivered to the host cell or host animal via aviral vector.

The invention also provides a recombinant host cell which produces anantibody as disclosed herein, optionally wherein the host cell comprisesthe isolated nucleic acid(s) or vector(s) according to the presentinvention. Typically, the host cell has been transformed or transfectedwith the nucleic acid(s) or vector(s). The recombinant host cell ofclaim can be, for example, a eukaryotic cell, a prokaryotic cell, or amicrobial cell, e.g., a transfectoma. In a particular embodiment thehost cell is a eukaryotic cell. In a particular embodiment the host cellis a prokaryotic cell. In some embodiments, the antibody is aheavy-chain antibody. In most embodiments, however, the antibody willcontain both a heavy and a light chain and thus said host cell expressesboth heavy- and light-chain-encoding construct, either on the same or adifferent vector.

Examples of host cells include yeast, bacterial, plant and mammaliancells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, NS0cells, Sp2/0 cells or lymphocytic cells. In one embodiment the host cellis a CHO (Chinese Hamster Ovary) cell. For example, in one embodiment,the host cell may comprise a first and second nucleic acid constructstably integrated into the cellular genome, wherein the first encodesthe heavy chain and the second encodes the light chain of an antibodyvariant as disclosed herein. In another embodiment, the presentinvention provides a cell comprising a non-integrated nucleic acid, suchas a plasmid, cosmid, phagemid, or linear expression element, whichcomprises a first and second nucleic acid construct as specified above.

In one embodiment, said host cell is a cell which is capable ofAsn-linked glycosylation of proteins, e.g. a eukaryotic cell, such as amammalian cell, e.g. a human cell.

In one embodiment, said host cell is a host cell which is not capable ofefficiently removing C-terminal lysine K447 residues from antibody heavychains. For example, Table 2 in Liu et al. (2008) J Pharm Sci 97: 2426(incorporated herein by reference) lists a number of such antibodyproduction systems, e.g. Sp2/0, NS/0 or transgenic mammary gland (goat),wherein only partial removal of C-terminal lysines is obtained. In oneembodiment, the host cell is a host cell with altered glycosylationmachinery. Such cells have been described in the art and can be used ashost cells in which to express variants of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al.(1999) Nat. Biotech. 17:176-1, as well as EP1176195; WO03/035835; andWO99/54342. Additional methods for generating engineered glycoforms areknown in the art, and include but are not limited to those described inDavies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002,J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem278:3466-3473), US6602684, WO00/61739A1; WO01/292246A1; WO02/31114OA1;WO 02/30954A1; Potelligent™ technology (Biowa, Inc. Princeton, N.J.);GlycoMAb™ glycosylation engineering technology (GLYCART biotechnologyAG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB,336: 1239-49, as well as those described in WO2018/114877 WO2018/114878and WO2018/114879.

In an even further aspect, the invention relates to a transgenicnon-human animal or plant comprising nucleic acids encoding one or twosets of a human heavy chain and a human light chain, wherein the animalor plant produces an antibody as disclosed herein.

In one embodiment, there is provided an antibody obtained or obtainableby the method described above.

In another aspect, the present invention also relates to a method ofincreasing or decreasing at least one effector function of an antibodyof the invention comprising introducing a mutation into the antibody inone or more amino acid residue(s) corresponding to E430, E345, and P329in the Fc region of a human IgG1 heavy chain, numbered according to theEu-index.

So, in certain embodiments, there is provided a method of increasing aneffector function of a parent antibody, such as an Fc-mediated effectorfunction or such as increasing the biological activity of the antibody,such as CD27 agonism, said parent antibody comprising an Fc region andan antigen-binding region binding to CD27, which method comprisesintroducing into the Fc region a mutation in one or both amino acidresidues corresponding to E430 and E345 in the Fc region of a human IgG1heavy chain, wherein the amino acid residues are numbered according tothe Eu index; and wherein the antigen-binding region comprises a VH CDR1comprising the sequence as set forth in SEQ ID NO:5, a VH CDR2comprising the sequence as set forth in SEQ ID NO:6, a VH CDR3comprising the sequence as set forth in SEQ ID NO:7, a VL CDR1comprising the sequence as set forth in SEQ ID NO:9, a VL CDR2comprising the sequence as set forth in SEQ ID NO:10, and a VL CDR3comprising the sequence as set forth in SEQ ID NO:11.

In other certain embodiments, there is provided a method of decreasingan effector function, such as C1q binding or FcgR binding, of a parentantibody comprising a VH CDR1 comprising the sequence as set forth inSEQ ID NO:5, a VH CDR2 comprising the sequence as set forth in SEQ IDNO:6, a VH CDR3 comprising the sequence as set forth in SEQ ID NO:7, aVL CDR1 comprising the sequence as set forth in SEQ ID NO:9, a VL CDR2comprising the sequence as set forth in SEQ ID NO:10, and a VL CDR3comprising the sequence as set forth in SEQ ID NO:11 and furthercomprising an amino acid substitution of E345R in the Fc region of ahuman IgG1 heavy chain, wherein the amino acid residues are numberedaccording to the Eu index, the method comprising introducing a furtheramino acid substitution in the Fc region at the amino acid positioncorresponding to P329 of a human IgG1 heavy chain, numbered according tothe Eu-index. In a preferred embodiment of the invention the methodcomprises the substitution of P329R. Hereby an effector function of theparent antibody, such as C1q binding or FcgR binding may be decreased ormay be completely eliminated.

In one embodiment of any one of the aforementioned methods, the effectorfunction which is increased comprises CD27 agonism.

In one embodiment of any one of the aforementioned methods, the effectorfunction is C1q binding.

In one embodiment of any one of the aforementioned methods, the effectorfunction is FcgR binding.

In one embodiment of any one of the aforementioned methods, the effectorfunctions that are decreseased comprises both C1q- and FcgR binding.

In one embodiment of any of the aforementioned methods, the mutation inthe one or more amino acid residues is selected from the groupcomprising: E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y andP329K. For example, the mutation in the one or more amino acidresidue(s) may comprise or consist of E430G or E345R.

In one embodiment of any of the aforementioned methods, the Fc region ofthe antibody is, apart from the recited mutation(s), a human IgG1, IgG2,IgG3 or IgG4 Fc region, or an isotype mixture thereof. Optionallycomprising an Fc region of one of the sequences set forth as SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, , SEQ ID NO:23, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:36. In a particularembodiment, the Fc region of the antibody is a human IgG1 Fc region. Forexample, the antibody can be a human full-length IgG1 antibody,optionally a human monoclonal full-length bivalent IgG1,κ antibody.Additionally, the antibody can be a monospecific or bispecific antibody,such as a monospecific antibody.

While the Fc region of the antibody may be a naturally occurring(wild-type) sequence, in some embodiments, the Fc region of the antibodycomprises one or more further mutations, as described elsewhere herein.

The present invention also relates to an antibody obtained or obtainableaccording to any of the above described methods.

The present invention also relates to a composition comprising anantibody according to the present invention, a nucleic acid according tothe present invention, an expression vector according to the presentinvention or a host cell according to the present invention.

In a further embodiment the composition according to the presentinvention is a pharmaceutical composition, typically comprising apharmaceutically acceptable carrier. In one embodiment thepharmaceutical composition contains an antibody as defined in any aspector embodiment disclosed herein, or an expression vector as defined inany aspect or embodiment disclosed herein.

In yet a further embodiment, the invention relates to a pharmaceuticalcomposition comprising:

-   an antibody as defined in any of the aspects and embodiments    disclosed herein, and-   a pharmaceutically acceptable carrier.

In one embodiment the pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

The invention also relates to kit-of-parts, such as a kit for use as acompanion diagnostic for identifying within a population of patientsthose patients which have a propensity to respond to treatment with anantibody as defined herein, comprising an antibody as defined in anyaspect or embodiment disclosed herein; and instructions for use of saidkit.

The invention also relates to kit-of-parts for use in therapy comprisingan antibody according to the invention, or a composition comprising anantibody according to the invention, optionally wherein the kit-of-partscontains more than one dosage of the antibody.

In one embodiment, the kit-of-parts comprises such an antibody orcomposition in one or more containers such as vials.

In one embodiment, the kit-of-parts comprises such an antibody orcomposition for simultaneous, separate or sequential use in therapy.

The antibodies of the present invention have numerous therapeuticutilities involving the treatment of diseases and disorders that may betreated by activating immune cells expressing CD27. For example, theantibodies may be administered to cells in culture, e.g., in vitro or exvivo, or to human subjects, e.g., in vivo, to treat or prevent a varietyof disorders and diseases. As used herein, the term “subject” isintended to include human and non-human animals which may benefit orrespond to the antibody. Subjects may for instance include humanpatients having diseases or disorders that may be corrected orameliorated by modulating CD27 function so that e.g. CD4⁺ and/or CD8⁺T-cell populations are expanded. Accordingly, the antibodies may be usedto elicit in vivo or in vitro proliferation of T-cell populations suchas T-helper cells and cytotoxic T-cells.

Thus, in one aspect, the present invention relates to the antibodiesaccording to the present invention, the nucleic acid or combination ofnucleic acids according to the present invention, the delivery vehicleaccording to the present invention, the expression vector according tothe present invention, the host cell according to the present invention,the composition according to the present invention, or thepharmaceutical composition according to the present invention for use asa medicament.

In one aspect, the present invention relates to the use of theantibodies according to the present invention, the nucleic acid orcombination of nucleic acids according to the present invention, thedelivery vehicle according to the present invention, the expressionvector according to the present invention, the host cell according tothe present invention, the composition according to the presentinvention, or the pharmaceutical composition according to the presentinvention in the preparation of a medicament for treating or preventinga disease or disorder.

In one aspect, the present invention relates to a method of treatment ofa disease or disorder comprising administering the antibody according tothe present invention, the nucleic acid or combination of nucleic acidsaccording to the present invention, the delivery vehicle according tothe present invention, the expression vector according to the presentinvention, the host cell according to claim the present invention, thecomposition according to the present invention, or the pharmaceuticalcomposition according to the present invention to a subject in needthereof.

In one aspect, the invention relates to the antibody according to anyaspect or embodiment for use as a medicament.

In one aspect, the invention relates to the use of the antibodyaccording to any aspect or embodiment in the preparation of a medicamentfor treating or preventing a disease or disorder.

In one aspect, the invention relates to the antibody according to anyaspect or embodiment for use in the treatment or prevention of a diseaseor disorder.

In one aspect, the invention relates to the antibody according to anyaspect or embodiment for use in diagnostic or for use in a diagnosticmethod.

In one aspect, the invention relates to a method of treating a diseaseor disorder, comprising administering the antibody according to anyaspect or embodiment to a subject in need thereof, typically in atherapeutically effective amount and/or for a time sufficient to treatthe disease or disorder.

In one aspect, the invention relates to a pharmaceutical compositioncomprising the antibody according to any aspect or embodiment, for useas a medicament.

In one aspect, the invention relates to a pharmaceutical compositioncomprising the antibody according to any aspect or embodiment for use inthe treatment or prevention of a disease or disorder.

In one aspect, the invention relates to a method of treatment of adisease or disorder comprising administering a pharmaceuticalcomposition comprising the antibody according to any aspect orembodiment to a subject in need thereof, typically in a therapeuticallyeffective amount and/or for a time sufficient to treat the disease ordisorder.

In one aspect, the present invention relates to a method of treating adisease or disorder, comprising the steps of:

-   selecting a subject suffering from the disease or disorder, and-   administering to the subject the antibody according to any aspect or    embodiment, or a pharmaceutical composition comprising the antibody,    typically in a therapeutically effective amount and/or for a time    sufficient to treat the disease or disorder.

In one embodiment, the disease or disorder is cancer, i.e. a tumorigenicdisorder, such as for example, a hematological cancer or a solid tumormalignancy. In another embodiment the disease or disorder is aninflammatory and/or autoimmune disease or disorder.

In a further aspect, the invention relates to an anti-idiotypic antibodywhich binds to an antibody comprising at least one antigen-bindingregion capable of binding to CD27, i.e. an antibody according to theinvention as described herein. In particular embodiments, theanti-idiotypic antibody binds to the antigen-binding region capable ofbinding to CD27 as described herein.

An anti-idiotypic (Id) antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody. An anti-Id antibody may be prepared by immunizing an animal ofthe same species and genetic type as the source of an anti-CD27monoclonal antibody with the monoclonal antibody against which ananti-Id is being prepared. The immunized animal typically can recognizeand respond to the idiotypic determinants of the immunizing antibody byproducing an antibody to these idiotypic determinants (the anti-Idantibody). A method for producing such antibodies is described in forinstance US 4,699,880. Such antibodies are further features of thepresent invention.

An anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. An anti-anti-ld antibody may be epitopicallyidentical to the original monoclonal antibody, which induced the anti-Idantibody. Thus, by using antibodies to the idiotypic determinants of amonoclonal antibody, it is possible to identify other clones expressingantibodies of identical specificity. Anti-Id antibodies may be varied(thereby producing anti-Id antibody variants) and/or derivatized by anysuitable technique, such as those described elsewhere herein withrespect to CD27-specific antibodies of the present invention. Forexample, a monoclonal anti-Id antibody may be coupled to a carrier suchas keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice.Sera from these mice typically will contain anti-anti-ld antibodies thathave the binding properties similar, if not identical, to anoriginal/parental anti-CD27 antibody.

Fc regions may have at their C-terminus a lysine. The origin of thislysine is a naturally occurring sequence found in humans from whichthese Fc regions are derived. During cell culture production ofrecombinant antibodies, this terminal lysine can be cleaved off byproteolysis by endogenous carboxypeptidase(s), resulting in a constantregion having the same sequence but lacking the C-terminal lysine. Formanufacturing purposes of antibodies, the DNA encoding this terminallysine can be omitted from the sequence such that antibodies areproduced without the lysine. Antibodies produced from nucleic acidsequences that either do, or do not encode a terminal lysine aresubstantially identical in sequence and in function since the degree ofprocessing of the terminal lysine is typically high when e.g. usingantibodies produced in CHO-based production systems (Dick, L.W. et al.Biotechnol. Bioeng. 2008;100: 1132-1143).

Hence, it is understood that proteins in accordance with the invention,such as antibodies, can be generated with or without encoding or havinga terminal lysine. It is also understood in accordance with theinvention that, sequences with a terminal lysine, such as a constantregion sequence having a terminal lysine, can be understood as thecorresponding sequences without a terminal lysine, and that sequenceswithout a terminal lysine can also be understood as the correspondingsequences with a terminal lysine.

EXAMPLES Example 1: Generation of Anti-Human CD27 Antibodies and FcVariants Thereof

Generation of anti-human CD27 antibodies through immunization andhybridoma generation was performed at Aldevron GmbH (Freiburg, Germany).cDNA’s encoding human CD27 (full length and ECD) were cloned intoAldevron proprietary expression plasmids. Anti-CD27 antibodies weregenerated by immunization of OmniRat animals (transgenic rats expressinga diversified repertoire of antibodies with fully human idiotypes;Ligand Pharmaceuticals Inc.) using intradermal application of human CD27cDNA-coated gold-particles using a hand-held device forparticle-bombardment (“gene gun”). Serum samples were collected after aseries of immunizations and tested by flow cytometry on HEK cellstransiently transfected with the aforementioned expression plasmid forfull length human CD27 expression. Antibody-producing cells wereisolated from rat spleen and fused with mouse myeloma cells (Ag8)according to standard procedures. RNA from hybridomas producingCD27-specific antibody was extracted for sequencing.

Out of a panel of 71 CD27 antibodies six antibodies were selected forfurther characterization based on binding to primary T cells anddiversity in CD27 binding competition assays in vitro. These sixantibodies are named IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D,IgG1-CD27-E and IgG1-CD27-F herein.

The variable regions, in some cases with single point mutations toremove amino acid residues that were considered a liability formanufacturing (e.g., free cysteines or glycosylation sites), of heavyand light chains of interest were gene synthesized and cloned intoexpression vectors containing the backbone sequences for human antibodylight chains and a human IgG1 heavy chain.

Fc variants of the six different antibodies were generated byintroduction of one or more of the following amino acid mutations,according to Eu numbering: E345R, E430G, P329R, G237A, K326A, E333A, seeTables 3 and 5 below. After functional characterization in vitro asdescribed below, CD27-specific IgGl-CD27-A was considered to have themost optimal biological properties. Sequences of the prior artCD27-targeting antibodies used herein as benchmarks have been obtainedas follows: IgG1-CD27-15 (WO2012004367; SEQ ID Nos 3 and 4),IgG1-CD27-131A (WO2018/058022; SEQ ID Nos 10 and 15), IgG1-CD27-CDX1127(WO2016145085; SEQ ID Nos: 1 and 2), and IgG1-CD27-BMS986215(WO2019195452A1; SEQ ID Nos 8 and 9). The VH and VL sequences of a typeI anti-human CD20 antibody have been described previously inWO2019/145455A1 (SEQ ID Nos 35 and 39).

TABLE 3 list of amino acid sequences SEQ ID NO: Identifier Domain Aminoacid sequence Organism 1 Human CD27 ORFMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPHomo sapiens 2 CD27-A59T variant ORFMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKTAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP Homo sapiens 3 Cyno CD27 ORFMARPHPWWLCFLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCHPCIPGVSFSPDHHTRPHCESCRHCNSGLLIRNCTITANAVCACRNGWQCRDKECTECDPPPNPSLTTWPSQALGPHPQPTHLPYVNEMLEARTAGHMQTLADFRHLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGTLFLHQQRKYRSNKGESPMEPAEPCPYSCPREEEGSTIPIQEDYRKPEPASSPMacaca fascicularis 4 CD27-A VH VHQVQLMQSGSELKKPGASVKVSCRASGYTFTTYAMNWVRQAPGQGPEWMGWINTNTGNPTYAQGFTGRFVFSLDTTVTTTYLQISSLKAEDTAVYFCAREAGSFDYWGQGTLVTVSSsynthetic construct 5 CD27-A VH CDR1 VH_CDR1 GYTFTTYA syntheticconstruct 6 CD27-A VH CDR2 VH_CDR2 INTNTGNP synthetic construct 7 CD27-AVH CDR3 VH_CDR3 AREAGSFDY synthetic construct 8 CD27-A VL VLQSALTQPASVSGSPGQSITISCTGTSSDVYYYNYVSWYQQHPGRAPKLVIYDVSNRPSGVSNRFSGSKSGNTASLTISGLRAEDEADYYCSSYTVNRVWVFGGGTKLTVLsynthetic construct 9 CD27-A VL CDR1 VL_CDR1 SSDVYYYNY syntheticconstruct 10 CD27-A VL CDR2 VL_CDR2 DVS synthetic construct 11 CD27-A VLCDR3 VL_CDR3 SSYTVN RVWV synthetic construct 12 Constant region humanIgG1m(f) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsynthetic construct 13 Constant region human IgG1-E345R ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsynthetic construct 14 Constant region human IgG1-P329R ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALRAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsynthetic construct 15 Constant region human IgG1-delK E345R+P 329RConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALR APIEKTISKAKGQPR RPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 16 Human kappa LC ConstantRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECsynthetic construct 17 Human lambda LC ConstantGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSsynthetic construct 18 Constant region human lgG1m(f) -delK ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 19 Constant region human IgG1m(a ) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKPVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsynthetic construct 20 Constant region human IgG1m(x ) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKPVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGKsynthetic construct 21 Constant region human IgG2 ConstantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsynthetic construct 22 Constant region human IgG3 ConstantASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKsynthetic construct 23 Constant region human IgG4 ConstantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKsynthetic construct 24 CD27-A IgG1 HC with E345R+P 329R Heavy chainconstant + VHQVQLMQSGSELKKPGASVKVSCRASGYTFTTYAMNWVRQAPGQGPEWMGWINTNTGNPTYAQGFTGRFVFSLDTTVTTTYLQISSLKAEDTAVYFCAREAGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALRAPIEKTISKAKGQPRRPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsynthetic construct 25 CD27-A LC Light chain constant + VLQSALTQPASVSGSPGQSITISCTGTSSDVYYYNYVSWYQQHPGRAPKLVIYDVSNRPSGVSNRFSGSKSGNTASLTISGLRAEDEADYYCSSYTVNRVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSsynthetic construct 26 Mouse CD27 ORFMAWPPPYWLCMLGTLVGLSATLAPNSCPDKHYWTGGGLCCRMCEPGTFFVKDCEQDRTAAQCDPCIPGTSFSPDYHTRPHCESCRHCNSGFLIRNCTVTANAECSCSKNWQCRDQECTECDPPLNPALTRQPSETPSPQPPPTHLPHGTEKPSWPLHRQLPNSTVYSQRSSHRPLCSSDCIRIFVTFSSMFLIFVLGAILFFHQRRNHGPNEDRQAVPEEPCPYSCPREEEGSAIPIQEDYRKPEPAFYPMus musculus 27 Human IgG1-Fc-E345R+P 329R ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALR APIEKTISKAKGQPR RPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsynthetic construct 28 Constant region human IgG1m(f) (withoutc-terminal lysine) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 29 Constant region human IgG1-E345R (withoutc-terminal lysine) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 30 Constant region human IgG1-P329R (withoutc-terminal lysine) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALRAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 31 Constant region human IgGlm(a) (withoutc-terminal lysine) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKPVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 32 Constant region human lgG1m(x) (withoutc-terminal lysine) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKPVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGsynthetic construct 33 Constant region human IgG2 (without c-terminallysine) ConstantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 34 Constant region human IgG3 (without c-terminallysine) ConstantASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDlAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGsynthetic construct 35 CD27-A IgG1 HC with E345R+P 329R (withoutc-terminal lysine) Heavy chain constant + VHQVQLMQSGSELKKPGASVKVSCRASGYTFTTYAMNWVRQAPGQGPEWMGWINTNTGNPTYAQGFTGRFVFSLDTTVTTTYLQISSLKAEDTAVYFCAREAGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALR APIEKTISKAKGQPR RPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 36 Human IgG1-Fc-E345R+P 329R (without c-terminallysine) ConstantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALRAPIEKTISKAKGQPRRPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGsynthetic construct 37 IgG1-CD27-A-P329RE345R-LNluc LC LCQSALQPASVSGSPGQSITISCTGTSSDVYYYNYVSWYQQHPGRAPKLVIYDVSNRPSGVSNRFSGSKSGNTASLTISGLRAEDEADYYCSSYTVNRVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSLGSSGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAsynthetic construct 38 IgG1-CD27-A-P329RE345R-LHalo LC LCQSALTQPASVSGSPGQSITISCTGTSSDVYYYNYVSWYQQHPGRAPKLVIYDVSNRPSGVSNRFSGSKSGNTASLTISGLRAEDEADYYCSSYTVNRVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGsynthetic construct 39 IgG1-b12-P329RE345R-LNLuc LC LCEIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSLGSSGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVRGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAsynthetic construct 40 IgG1-b12-P329R-E345R-LHalo LC LCEIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGsynthetic construct 41 IgG1-CD27-A-LNLuc LC LCQSALTQPASVSGSPGQSITISCTGTSSDVYYYNYVSWYQQHPGRAPKLVIYDVSNRPSGVSNRFSGSKSGNTASLTISGLRAEDEADYYCSSYTVNRVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSLGSSGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAsynthetic construct 42 IgG1-CD27-A-LHalo LC LCQSALTQPASVSGSPGQSITISCTGTSSDVYYYNYVSWYQQHPGRAPKLVIYDVSNRPSGVSNRFSGSKSGNTASLTISGLRAEDEADYYCSSYTVNRVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGsynthetic construct 43 IgG1-CD20-11B8-E430G-LNLuc LC LCEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSLGSSGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAsynthetic construct 44 IgG1-CD37-37.3-E430G-LHalo LC LCDIQMTQSPASLSVSVGETVTITCRASENlRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGsynthetic construct

Example 2: Agonist Activity of Anti-CD27 Antibodies in a CD27 ActivationReporter Cell Assay

CD27 agonist activity of the different anti-CD27 antibodies with andwithout an E345R or an E430G hexamerization-enhancing Fc mutation wasmeasured using the CD27 Thaw and Use Bioassay kit (Promega, Custom AssayServices, CAS # CS1979A25). The kit contains NF-κB Reporter-Jurkatrecombinant cells expressing the firefly luciferase gene under thecontrol of NF-κB response elements with constitutive expression of humanCD27 and was used essentially according to the manufacturer’sinstructions. Briefly, Thaw-and-Use GloResponse NFκB-luc2/CD27 Jurkatcells were thawed and incubated in 96-well flat bottom culture plates(PerkinElmer, Cat # 6005680) with antibody dilution series (finalconcentration range 0.04 - 20 µg/mL) in Bio-Glo Luciferase Assay Bufferfor 6h at 37° C., 5% CO₂. The anti-CD27 antibodies were wild-type (WT*)IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E,IgG1-CD27-F, and variants of each one harboring the E430G or E345Rmutation. Anti-CD27 benchmark antibodies were IgG1-CD27-131A (WT andE430G variant) and a non-hexamerizing IgG1-CD27-15(IgG1-CD27-15-P329RE345R-K439E, that carries a combination of Fcmutations that prevents hexamerization and thus the mutations arefunctionally irrelevant in the context of this experiment and istherefore referred to as WT in the figure) and a hexamerizing variant ofIgG1-CD27-15 comprising a E345R mutation. An anti-HIV gp120 humanantibody, IgG1-b12-E345R, was used as a non-binding negative controlantibody (ctrl). After the antibody incubation, Bio-Glo Luciferase AssayReagent (equilibrated to RT) was added to each well and incubated at RTfor 5-10 min. Luminescence was measured using an EnVision MultilabelReader (PerkinElmer) and presented as relative luminescence units (RLU)in bar diagrams generated using GraphPad Prism software.

Introduction of a hexamerization-enhancing Fc mutation (E345R or E430G)resulted in enhanced CD27 agonism compared to the corresponding WTantibody for antibody clones IgG1-CD27-A to -E and for the benchmarkantibodies IgG1-CD27-131A (tested with E430G) and IgG1-CD27-15 (testedwith E345R) (FIG. 1 ).

Whereas IgG-CD27-A, B and C demonstrated enhanced CD27 agonist activityafter introduction of E430G or E345R at all concentrations tested,IgG1-CD27-D and E variant containing hexamerization-enhancing mutationsdid not show increased agonism at the lowest antibody concentrations.IgG1-CD27-F variants with the E430G or E345R mutations only showedenhanced CD27 agonism at the highest antibody concentration tested. Forvariants IgG1-CD27-A to -E, introduction of the E345R mutation resultedin stronger CD27 activation than the E430G mutation. AntibodiesIgG1-CD27-A to -E having the E345R mutation showed higher or similarCD27 activation levels compared to IgG1-CD27-131A having the E430Gmutation or CD27-15 having the E345R mutation, respectively.

*The WT antibodies for IgG1-CD27-B and IgG1-CD27-F carried a F405Lmutation in the IgG Fc domain, which is functionally irrelevant in thecontext of this experiment.

Example 3 Binding Affinities of Anti-Human CD27 Antibodies forRecombinant Human, Mouse And Cynomolgus Monkey CD27

The binding affinities of five anti-human CD27 IgG1 antibodies(IgG1-CD27-A, -B, -C, -D and -E) for recombinant human, cynomolgusmonkey and mouse CD27 protein were determined using label-free biolayerinterferometry on an Octet HTX instrument (FortéBio, Portsmouth, UK).Experiments were performed using bispecific antibodies comprising oneCD27-specific Fab-arm and a non-binding Fab-arm, so that the antibody ismonovalent for CD27. These bispecific antibodies were generated bycontrolled Fab-arm exchange between the CD27 antibodies and non-bindingantibodies (as described in Labrijn AF et al., Nat Protoc. 2014Oct;9(10):2450-63).

To determine the affinity of the CD27 antibodies for human and mouseCD27, 100 nM recombinant His-tagged mouse or human CD27 protein (SinoBiological, Cat # 10039-H08B1 [human], Cat # 50110-M08H [mouse]) wasloaded to pre-conditioned anti-Penta-HIS (HIS1K) biosensors (FortéBio,Cat # 18-5120) for 600 sec.

To assess the affinity of the CD27 antibodies for cynomolgus monkeyCD27, 5 µg/mL of recombinant cynomolgus monkey CD27-Fc fusion protein(R&D systems, Cat # 9904-CD-100) was loaded to activated Amine Reactive2nd Generation (AR2G) biosensors (FortéBio, Cat # 18-5092).

After baseline measurements in Sample Diluent (FortéBio, Cat # 18-1104)for 300 sec, the association (200 sec) and dissociation (1,000 sec) ofCD27 antibodies was determined for antibody concentration series of0.78-800 nM with two-fold dilution steps in Sample Diluent. An antibodymolecular mass of 150 kDa was used for calculations. Reference sensorswere incubated with Sample Diluent.

Data were acquired using Data Acquisition Software v11.1.1.19 (ForteBio)and analyzed with Data Analysis Software v9.0.0.14 (ForteBio). Datatraces were corrected per antibody by subtraction of the referencesensor. The Y-axis was aligned to the last 10 sec of the baseline andInterstep Correction alignment to dissociation and Savitzky-Golayfiltering were applied. Data traces were excluded from analysis when theresponse was < 0.05 nm and calculated equilibrium was near to saturation(Req/Rmax > 95% using a dissociation time of 50 sec). The data wasfitted with the 1:1 model using a window of interest for the associationset at 200 sec and dissociation time set at 50 sec. The dissociationtime was chosen based on the coefficient of determination (R²), which isan estimate of the goodness of the curve fit (preferentially > 0.98),visual inspection of the curve, and at least 5% signal decay during theassociation step.

Affinities for human CD27 could be accurately determined for three CD27antibodies (IgG1-CD27-A,B,C) with K_(D) values in the nanomolar range(Table 4). For IgG1-CD27-D, and -E, BioLayer Interferometry experimentsconfirmed binding to human CD27 with affinities in a similar range,although suboptimal curve fitting did not allow calculation of accurateK_(D) values (as indicated in Table 4).

IgG1-CD27-A and -B also showed binding to recombinant cynomolgus monkeyCD27, with K_(D) values in the same range as for human CD27. Resultsobtained with IgG1-CD27-C, -D and -E also confirmed binding tocynomolgus monkey CD27 with affinities in a similar range, althoughsuboptimal curve fitting did not allow calculation of accurate K_(D)values (as indicated in Table 4).

Binding to recombinant mouse CD27 was only observed for antibodyIgG1-CD27-C.

TABLE 4 Binding affinities of IgG1-CD27-A to -E antibodies to CD27 fromthe indicated species. Sample Loading sample K_(D)(M) k_(on) (1/Ms)k_(dis) (1/s) IgG1-CD27-A Human CD27-His 1.3E-07 1.3E+05 1.8E-02 MouseCD27-His n.b. Cyno CD27-Fc 1.2E-07 1.7E+05 2.0E-02 IgG1-CD27-B HumanCD27-His 5.4E-08 3.3E+05 1.8E-02 Mouse CD27-His n.b. Cyno CD27-Fc3.5E-08 6.5E+05 2.3E-02 IgG1-CD27-C Human CD27-His 7.0E-08 1.4E+059.8E-03 Mouse CD27-His 4.3E-07 5.0E+04 2.2E-02 Cyno CD27-Fc 5.1E-08*1.2E+05* 6.4E-03* IgG1-CD27-D Human CD27-His 4.5E-08* 1.4+05* 6.4E-03*Mouse CD27-His n.b. n.b. n.b. Cyno CD27-Fc 2.1E-08* 2.4E+05* 5.0E-03*IgG1-CD27-E Human CD27-His 4.9E-08* 1.3+05* 6.4E-03* Mouse CD27-His n.b.n.b. n.b. Cyno CD27-Fc 3.5E-08* 1.6E+05* 5.5E-03* * : binding wasobserved but KD, k_(on) and k_(dis) are less reliable values due tosuboptimal curve fitting, resulting in unreliable interpretation usingthe 1:1 model. n.b.: no binding observed.

Example 4: Binding of Anti-CD27 Antibodies to Cell Surface-ExpressedHuman and Cynomolgus Monkey CD27

Binding of anti-CD27 antibodies IgG1-CD27-A to -E* and prior artIgG1-CD27-131A* to cell surface-expressed human and cynomolgus monkeyCD27 was analyzed by flow cytometry using transiently transfectedHEK293F cells and primary T cells, which endogenously express CD27.Non-binding control antibody IgG1-b12-FEAR was used as negative controlantibody.

FreeStyle 293-F suspension cells (HEK293F; ThermoFisher, Cat # R79007)were transiently transfected with mammalian expression vector pSBencoding full length human or cynomolgus monkey CD27 using 293fectinTransfection Reagent (ThermoFisher, Cat # 12347019) according to themanufacturer’s instructions.

Human and cynomolgus monkey PBMC were purified from buffy coats obtainedfrom human healthy donors (Sanquin Blood Bank, the Netherlands) or froma cynomolgus monkey (BPRC, the Netherlands, Cat # S-1135) by low densitygradient centrifugation using Lymphocyte Separation Medium (LSM;Corning, Cat # 25-072CV) according to the manufacturer’s instructions.

Cells were seeded in 96-Wells plates (100,000 cells per well; GreinerBio-one, Cat # 650180) for sequential incubations, with washing steps inbetween with FACS buffer, consisting of PBS (Lonza, Cat # BE17-517Q) +1% BSA (Roche, Cat # 10735086001) + 0.02% Sodium Azide (Bio-World, Cat #41920044-3). The following incubations were applied: antibodyconcentration series (0.0001 - 10 µg/mL final concentration) for 30 minat 4° C.; live/dead marker FVS510 (BD, Cat # 564406, diluted 1:1,000 inPBS) for 20 min at RT; PE-labeled polyclonal goat anti-human IgG(Jackson Immuno Research, Cat # 109-116-098, diluted 1:500) for 30 minat 4° C.; and anti-CD3 antibody for T-cell identification (anti-humanCD3: BD, Cat # 555335, diluted 1:10; anti-cyno CD3: Miltenyi, Cat #130-091-998, diluted 1:10) for 30 min at 4° C. All samples were analyzedon a FACSCelesta flow cytometer (BD) and FlowJo software. Data wereprocessed and visualized using GraphPad Prism.

All tested antibodies showed dose-dependent binding to human CD27, bothon human T cells and transfected HEK293F cells (FIGS. 2 A,B). Highestmaximal binding was observed for IgG1-CD27-B and IgG1-CD27-C compared tointermediate binding for IgG1-CD27-A and IgG1-CD27-131A, and low bindingfor IgG1-CD27-D and IgG1-CD27-E, with the differences being mostpronounced using human T-cells. For binding to cynomolgus money CD27 Tcells, highest binding was observed for IgG1-CD27-B, followed byIg1-CD27-131A and IgG1-CD27-A. Lower binding was observed forIgG1-CD27-D and -E, whereas IgG1-CD27-C showed minimal binding tocynomolgus monkey T cells. All CD27 antibodies showed dose-dependentbinding to HEK cells transfected with cynomolgus monkey CD27. Highestmaximal binding was observed for IgG1-CD27-B and IgG1-CD27-131-A,somewhat lower binding was observed for IgG1-CD27-A, -D and -E.IgG1-CD27-C showed the lowest binding to HEK cells transfected withcynomolgus monkey CD27 (FIGS. 2 C,D).

In conclusion, IgG1-CD27-A and IgG1-CD27-B showed dose-dependent bindingto human and cynomolgus monkey CD27 expressed endogenously on human orcynomolgus monkey T cells, and transiently expressed in transfected HEKcells. IgG1-CD27-A and IgG-CD27-131A showed comparable binding to humanT cells, whereas IgG1-CD27-B showed higher maximal binding.

*N.B. IgG1-CD27-A, -B, -C, -D and -E carried mutationsF405L-L234F-L235E-D265A in the IgG Fc domain, which are functionallyirrelevant in the context of this experiment. IgG1-CD27-131A carried afunctionally irrelevant F405L mutation in the IgG1 Fc domain.

Example 5: Binding of Anti-CD27 Antibodies to a Natural Human CD27-A59TVariant

Approximately 19% of the human population expresses a natural CD27variant harboring an A59T mutation in the extracellular domain (SEQ IDNO. 2). Binding to human CD27-A59T was tested by flow cytometry foranti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C* andbenchmark IgG1-CD27-131A. Non-binding antibody IgG1-b12-FEAL was used asa negative control antibody. Transiently transfected HEK293F cellsexpressing human CD27-A59T (15,000 cells per well) were incubated withconcentration series (0.0001 - 10 µg/mL using 10-fold dilution steps) ofprimary test antibodies IgG1-CD27-A to -C, non-binding control antibodyIgG1-b12 (ctrl), and the prior art benchmark IgG-CD27-131A, which hasbeen described previously to bind to CD27-A59T (WO2018/058022). Afterincubation, antibodies were PE-labeled with polyclonal goat anti-humanIgG. Binding was analyzed on a FACSCelesta flow cytometer (BD) andFlowJo software. Data were processed and visualized using GraphPad Prismv.8.

The tested anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C,and IgG1-CD27-131A showed dose-dependent binding toCD27-A59T-transfected HEK293F cells with similar binding curves amongthe different antibodies (Figure EXAMPLE 5).

*N.B. IgG1-CD27-A, -B and -C carried mutations F405L-L234F-L235E-D265Ain the IgG Fc domain, which are functionally irrelevant in the contextof this experiment. IgG1-CD27-131A carried a functionally irrelevantF405L mutation in the IgG1 Fc domain.

Example 6: Induction of Human T Cell Proliferation by Anti-CD27Antibodies

As enhanced IgG hexamerization through Fc-Fc interactions uponintroduction of the E345R or E430G mutation enhanced CD27 agonistactivity of anti CD27 antibodies (example 2), the capacity ofIgG1-CD27-A, IgG1-CD27-B, and IgG1-CD27-C antibody variants carrying theE430G or E345R mutations to increase proliferation of TCR activated Tcells was tested in vitro.

Additionally, Fc mutations that were reported to reduce binding to C1qand FcgR (G237A or P329R) or that enhance binding to C1q (K326A/E333Adouble mutation) were introduced to test their potential effect on CD27agonist activity of CD27 antibodies carrying the E345R or E430Gmutations. The K326A/E333A double mutation was previously shown toenhance C1q binding and to contribute to enhanced agonistic activity ofDR5-specific humanized IgG1 antibodies comprising an Fc-Fc interactionenhancing mutation (WO2018/146317A1). The mutations G237A, P329R, orK326A/E333A were introduced, in addition to E430G or E345R, toIgG1-CD27-A, IgG1-CD27-B and IgG1-C (Table 5) and their effect on T-cellproliferation was determined using human PBMC obtained from healthydonors (Sanquin Blood Bank, the Netherlands).

TABLE 5 Mutations in the Fc domain of antibodies IgG1-CD27-A,IgG1-CD27-B, or IgG1-CD27-C and their biological effect Fc mutationE430G E345R P329R G237A K326/E333 A Described effect Enhancedhexamerization Enhanced hexamerization Reduced C1q/FcgR binding ReducedC1q/FcgR binding Enhanced C1q binding Antibody* IgG1-CD27-X-E430G +IgG1-CD27-X-P329R-E430G + + IgG1-CD27-X-G237A-E430G + +IgG1-CD27-X-K326A-E333A-E430G + + IgG1-CD27-X-E345R +IgG1-CD27-X-P329R-E345R + + IgG1-CD27-X-G237A-E345R + +IgG1-CD27-X-K326A-E333A-E345R + + *X in IgG1-CD27-X, refers to IgG1-CD27clones IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27–C.

PBMCs were resuspended in PBS at a density of 5 × 10⁶ cells/mL andlabeled with CFSE using CellTrace CFSE Cell Proliferation Kit(Invitrogen, Cat # C34564; 1:10,000), according to the manufacturer’sinstructions. CFSE-labeled PBMCs (100,000 cells/well) were incubated in96-well round-bottom plates (Greiner Bio-one, Cat # 650180) with 0.1µg/mL anti-CD3 antibody clone UCHT1 (Stemcell Technologies, Cat # 60011)to activate T cells, and CD27 antibodies (1 µg/mL final concentration)in T-cell Activation Medium (ATCC, Cat # 80528190) supplemented with 5%Normal Human Serum (NHS; Sanquin, Product # B0625) for 96 h at 37° C./5%CO₂. For identification of viable cells in CD4⁺ and CD8⁺ T-cell subsetsby flow cytometry, cells were sequentially incubated with live/deadmarker FVS510 (1:1,000) for 20 min at RT and a staining mix forlymphocyte markers, containing APC-eFluor780-labeled anti-human CD4antibody (Invitrogen, Cat # 47-0048-42, 1:50), AlexaFluor700-labeledanti-human CD8a antibody (BioLegend, Cat # 301028; 1:100),PE-Cy7-labeled mouse anti-human CD14 antibody (BD Biosciences, Cat #557742; 1:50) and BV785-labeled anti-human CD19 antibody (BioLegend, Cat# 363028; 1:50) for 30 min at 4° C. in the dark. Samples were measuredon a FACSCelesta (BD Biosciences) flow cytometer and CFSE dilution peaksin the viable CD4⁺ and CD8⁺ T-cell subsets (FVS510⁻CD14⁻CD19⁻CD4⁺ andFVS510⁻CD14⁻CD19⁻CD8⁺) were analyzed using FlowJo 10 software as areadout for T-cell proliferation. T-cell proliferation was expressed asthe percentage of proliferated cells or the division index bothcalculated by using the FlowJo software (version 10). Percentage ofproliferated (divided) cells was determined by gating for the cells thathave gone through CFSE dilution (CFSE^(low) ^(peaks)). The divisionindex is the average number of divisions that the cells underwent.Heatmaps were generated using GraphPad Prism version 8. Proliferationassays were performed using PBMC from four different healthy donors.

Variants of IgG1-CD27-A, -B and -C carrying an E430G or E345R mutationinduced a small increase in proliferation of CD8⁺ T cells compared tocontrol antibody in two out of the four donors tested. The introductionof additional mutations (P329R, G237A or K326A/E333A) into IgG1-CD27-A,-B or -C variants carrying an E430G mutation showed variable effects onCD8⁺ T cell proliferation across the four PBMC donors. In contrast,introduction of the P329R mutation into IgG1-CD27-A and IgG1-CD27-Cvariants carrying an E345R mutation consistently increased theircapacity to enhance proliferation of activated CD8⁺T cells. Thisparticularly applied to IgG1-CD27-A: whereas the measured CD8⁺T cellproliferation was comparable for IgG-CD27-A-E345R, IgG1-CD27-B-E345R andIgG1-CD27-C-E345R in each of the donors, introduction of an additionalP329R mutation consistently led to a higher increase in CD8⁺ T cellproliferation for clone IgG1-CD27-A-E345R compared to IgG1-CD27-B-E345Ror IgG1-CD27-C-E345R. Thus, the effect of the E345R mutation incombination with the P329R mutation on proliferation of TCR activatedCD8⁺T cells was consistently larger for clone IgG1-CD27-A than forIgG1-CD27-B and IgG1-CD27-C. Across all antibody variants tested,IgG1-CD27-A-E345R-P329R induced the largest increase in CD8⁺T cellproliferation in all donors (FIG. 4A).

The addition of the mutations G237A or K326A-E333A into CD27 antibodyvariants carrying the E345R mutation did not or only minimally increasethe proliferation of CD8⁺ T cells in any of the clones tested, ascompared to antibodies comprising the single mutations E345R (FIG. 4A).

Also in CD4⁺ T cells, the highest and most consistent increase in T cellproliferation was observed in presence of IgG1-CD27-A-E345R-P329R.Whereas CD4⁺ T cell proliferation was generally comparable betweenIgG1-CD27-A, -B and -C variants carrying only the E430G or E345Rmutations, introduction of an additional P329R mutation led to a largerincrease in CD4⁺ T cell proliferation for the IgG1-CD27-A variantcarrying the E345R variant compared to IgG1-CD27-A-E430G or IgG1-CD27-Bor -C variants carrying either the E430G or the E345R mutation. Thiseffect was observed in three out of four donors tested. In donor 1, theeffect of additional mutations in addition to E430G or E345R on CD4⁺ Tcell proliferation was generally small, and effects observed in thisdonor were not reproduced in the other three donors.

The combination of the E345R with the P329R mutations also consistentlyincreased CD4⁺ T cell proliferation for IgG1-CD27-C, although thedifference between the E345R mutation alone and the combination of E345Rand P329R was smaller for clone IgG1-CD27-C than for clone -A. For cloneIgG1-CD27-B, a modest increase in CD4⁺ T cell proliferation was observedfor IgG1-CD27-B-E345R-P329R compared to IgG1-CD27-B-E345R in two out ofthe four donors.

Introduction of the P329R, G327A or K326A/E333A mutations intoIgG1-CD27-A, -B, or -C variants carrying the E430G mutation did not, ornot consistently, induce effects on CD4⁺ T cell proliferation.Similarly, no or inconsistent effects were observed after introductionof the G327A or K326A/E333A in IgG1-CD27-A, -B or -C variants carryingthe E345R mutation.

In summary, IgG1-CD27-A-E345R-P329R consistently induced the highestincrease in proliferation of activated CD8⁺ and CD4⁺ T cells,demonstrating that IgG1-CD27-A-E345R-P329R induces most efficient CD27agonism. DR5-specific, hexamerization-enhanced antibodies with the P329Rmutation previously showed reduced capacity to induce DR5 agonismcompared to DR5-specific hexamerization-enhanced antibodies without theP329R mutation (Overdijk et al, Mol Canc Ther 2020). It was thusconsidered surprising that introduction of the P329R mutation inaddition to the E345R mutation in IgG1-CD27-A enhanced CD27 agonistactivity. Moreover, it is not known why the combined effect of theE345R+P329R mutations was consistently larger for IgG1-CD27-A than forIgG1-CD27-B or IgG1-CD27-C.

Example 7: Induction of Human T-Cell Proliferation by Anti-CD27 AntibodyIgG1-CD27-A-P329R-E345R

The capacity of IgG1-CD27-A-P329R-E345R to increase proliferation of TCRstimulated human CD4⁺ and CD8⁺ T-cells was analyzed in CSFE dilutionassays using human healthy donor PBMC, and compared to prior artanti-CD27 clones IgG1-CD27-131A*, IgG1-CD27-CDX1127, andIgG1-CD27-BMS986215*. The T-cell proliferation assays were performed asdescribed in Example 6, with minor deviations (75,000 cells/well;concentration range 0.002 - 10 µg/mL). Samples using T-cells withoutanti-CD3 stimulation were included to test potential CD27 agonistactivity of the antibodies in absence of T-cell receptor activation(FIGS. 5A and 5B). Such activity is unwanted as it would pose a safetyrisk if the antibody was able to induce proliferation of resting Tcells.

Percentage of proliferated T cells (FIGS. 5A, B, C, D) was calculated asthe percentage of cells with reduced CFSE fluorescence, indicating celldivisions using FlowJo software. Expansion index (FIGS. 5E and 5F)identifies the fold increase of cells in the wells and was calculatedusing the Proliferation Modeling tool in FlowJo version 10. Manualadjustments to the peaks were made where necessary to define the numberof the peaks present more consistently.

None of the CD27 antibodies of the invention and the prior artantibodies tested here induced proliferation of unstimulated T cells(i.e., in absence of CD3 crosslinking (FIGS. 5A and B).

Most of the CD27 antibodies induced some proliferation of activated CD4⁺and CD8⁺ T-cells at the highest antibody concentrations tested (FIGS. 5Cand D). Based on this, an expansion index was calculated (FIGS. 5E andF). Antibody IgG1-CD27-A-P329R-E345R of the invention more profoundlyenhanced proliferation of CD4⁺ and CD8⁺ T cells in vitro compared to theprior art anti-CD27 clones IgG1-CD27-131A, IgG1-CD27-CDX1127 andIgG1-CD27-BMS986215.

*For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants carrying a F405Lmutation, that is functionally irrelevant in the context of thisexperiments, were used.

Example 8: C1q Binding to Membrane-Bound CD27 Antibodies

The P329R mutation was previously described to reduce interaction ofIgG1 antibodies with C1q and FcgR (Overdijk et al, Molecular CancerTherapeutics 2020). The effect of the P329R mutation on C1q binding ofIgG1-CD27-A comprising the E345R mutation was tested in cellular C1qbinding assays in vitro using human healthy donor T cells. Anti-HIVgp120 antibody IgG1-b12-F405L was used as non-binding isotype controlantibody (ctrl). T cells were enriched from human healthy donor PBMCsusing RosetteSep Human T cell Enrichment cocktail (Stemcell, Cat# 15061)and resuspended in culture medium (RPMI 1640 [Gibco, Cat # A10491-01]supplemented with 0.1% BSA and 1% Pen/Strep [Lonza, Cat # DE17-603E]). Tcells (2 x 10⁶ cells/well) were pre-incubated in polystyrene 96-wellround-bottom plates with antibody dilution series (8x five-fold dilutionstarting at 15 µg/mL final assay concentration) for 15 min at 37° C. toallow the antibodies to bind to the T cells. Then, cells were cooled onice, supplemented with NHS as a source of human C1q (20% NHS final assayconcentration) and incubated on ice for 45 min. Cells were subsequentlyincubated on ice with FITC-labeled Rabbit anti-human C1q antibody (DAKO,Cat # F0254; 20 µg/mL) for 30 min and resuspended in FACS buffer withTO-PRO-3 (ThermoFisher, Cat # T3605; 1:5,000 dilution). C1q binding wasdetermined by flow cytometry measuring the FITC signal on live cells.

Membrane bound WT IgG1-CD27-A antibody did not show C1q binding (FIG. 6). Introduction of the hexamerization-enhancing mutation E430G or E345R(IgG1-CD27-A-E430G and IgG1-CD27-A-E345R) resulted in binding of C1q toCD27 antibodies on the T-cell surface, in line with the increasedbinding avidity of the hexameric C1q protein to hexameric antibody ringstructures on the cell surface (FIG. 6 ). Introduction of the P329Rmutation in IgG1-CD27-A-E345R (IgG1-CD27-A-P329R-E345R) resulted in lossof C1q binding (FIG. 6 ), demonstrating that IgG1-CD27-A-P329R-E345R wasunable to bind C1q.

These data show that IgG1-CD27-A-P329R-E345R is unable to bind C1q uponbinding to CD27 on the cell surface of T cells. This indicates that C1qbinding does not contribute to antibody-induced CD27 agonist activity ofIgG1-CD27-A-P329R-E345R. This is in contrast to what was previouslydescribed for other hexamerization-enhanced agonistic antibodies.Moreover, lack of C1q binding indicates that IgG1-CD27-A-P329R-E345R isunable to activate the classical pathway of complement activation. Thus,IgG1-CD27-A-P329R-E345R is not expected to induce complement activationand CDC on T cells which activity would be unwanted.

Example 9: Binding of Anti-CD27 Antibodies to Human Fc Receptors

Binding of IgG1-CD27-A- P329R-E345R to human FcyR variants was analyzedusing a Biacore surface plasmon resonance (SPR) system and compared toan anti-HIV gp120 antibody IgG1-b12 (ctrl). Biacore Series S SensorChips CM5 (Cytiva, Cat # 29104988) were covalently coated with anti-Hisantibody using amine-coupling and His capture kits (Cytiva, Cat #BR100050 and Cat # 29234602) according to the manufacturer’sinstructions. Next, 125 nM Fcy-receptor FcyRla, FcyRlla (167-His [H] and167-Arg [R]), FcyRllb or FcyRllla (176-Phe [F] and 176-Val [V]) (SinoBiological, Cat # 10256-H08S-B, Cat # 10374-H27H, Cat # 10374-H27H1-B,Cat # 10259-H27H-B, Cat # 10389-H27H-B and Cat # 10389-H27H1-B) inHBS-P+

(Cytiva, Cat # BR100827) were captured onto the surface. After threecycles of buffer, antibody samples were injected for 36 cycles togenerate binding curves using antibody ranges of 0-3,000 nM for FcyRIand 0-10,000 nM for the other FcyRs. Each sample that was analyzed on anFcR-coated surface (Active Surface) was also analyzed on a parallelflow-cell without FcR (Reference Surface), which was used for backgroundcorrection. Dissociation from the anti-His-coated surface was performedby regeneration of the surface using 10 mM Glycine-HCl pH 1.5 (Cytiva,Cat # BR100354). Sensograms were generated using Biacore InsightEvaluation software (Cytiva) and a four-parameter logistic (4PL) fit wasapplied to calculate relative binding of IgG1-CD27-A-P329R-E345R againstthe reference sample (ctrl).

Binding of IgG1-CD27-A-P329R-E345R to high affinity receptor FcyRla wasstrongly reduced compared to the ctrl antibody, although some bindingwas observed at higher antibody concentrations (FIG. 7A). IgG1-CD27-A-P329R-E345R did not bind to the human low affinity receptors FcγRIIa(FIGS. 7B and C), FcγRIIb (FIG. 7D) and FcγRIIIa (FIGS. 7E and F).

In conclusion, IgG1-CD27A-P329R-E345R shows minimal (FcyRla) or no(FcyRlla, FcyRllb, and FcyRllla) binding to human IgG Fc receptors.

Example 10: Binding of Anti-CD27 Antibody IgG1-CD27-A-E345R-P329R toHuman T Cells

Binding of IgG1-CD27-A-P329R-E345R to CD27 on human healthy donor Tcells was characterized in more detail using flow cytometry. Anti-HIVgp120 antibody variant IgG1-b12- P329R- E345R was used as non-bindingcontrol antibody (ctrl). Human PBMCs were isolated from buffy coatsobtained from human healthy donors. PBMC (1 × 10⁵ cells/well) in FACSbuffer were added to polystyrene 96-well round-bottom plates (Greinerbio-one, Cat # 650101) and pelleted by centrifugation at 300×g for 3 minat 4° C. The cells were resuspended in 50 µL/well serial antibodydilutions in FACS buffer (range 0.0015 to 10 µg/mL in 3-fold dilutionsteps) and incubated for 30 min at 4° C. Cells were pelleted, washedtwice with FACS buffer and incubated in 50 µL/well with FITC-conjugatedsecondary antibody (FITC AffiniPure F(ab′)₂ fragment goat anti-humanIgG, F(ab′)₂ fragment specific, Jackson ImmunoResearch, Cat #109-096-097, diluted 1:100) for 30 min at 4° C. in the dark. Cells werepelleted again, washed twice with FACS buffer and incubated for 30 minat 4° C. in the dark in 50 µL/well of a staining mix for lymphocytemarkers, containing BV711-labeled anti-human CD19 antibody (BioLegend,Cat # 302246, 1:50), AlexaFluor700-labeled anti-human CD8a antibody(BioLegend, Cat # 301028, 1:100), APC-eFluor780-labeled anti-human CD4antibody (Invitrogen, Cat # 47-0048-42, 1:50), PE-CF594-labeled mouseanti-human CD56 antibody (BD Biosciences, Cat # 564849, 1:100),PE-Cy7-labeled mouse anti-human CD14 antibody (BD Biosciences, Cat #557742, 1:50) and eFluor450-labeled anti-human CD3 antibody (Invitrogen,Cat # 48-0037-42, 1:200).

Cells were pelleted again, washed twice using FACS buffer, andresuspended in 80 µL FACS buffer containing death cell marker7-Amino-Actinomycin D (7-AAD; BD Biosciences, Cat # 51-68981E, 1:240diluted). The samples were measured by flow cytometry on an LSRFortessa(BD) flow cytometer and analyzed using FlowJo software. Binding curveswere analyzed using non-linear regression (sigmoidal dose-response withvariable slope) using GraphPad Prism 8 software.

Anti-CD27 antibody IgG1-CD27-A-P329R-E345R showed dose-dependent bindingto healthy donor T cells, with similar binding characteristics for CD4⁺and CD8⁺ T cells (FIG. 8 ).

Example 11: FcyR-Independent Induction of CD27 Cell Signaling byAnti-CD27 Antibody IgG1-CD27-A-P329R-E345R

A CD27-specific monoclonal antibody that can induce CD27 signalingindependent of secondary FcyR-mediated cross-linking may beimmunostimulatory in the absence of FcyR-positive cells, which would bean advantage in tumors where the frequency of FcyR-bearing cells is low.

CD27 agonist activity of IgG1-CD27-A-P329R-E345R was tested in thepresence or absence of FcyR-bearing cells and compared to thecorresponding WT antibody IgG1-CD27-A and prior art antibodiesIgG1-CD27-131A*, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215*.Non-binding antibody IgG1-b12-P329R-E345R was used as a negative control(ctrl). CD27 reporter assays were performed, essentially as described inExample 2, with the exception that in the current example, Thaw-and-UseGloResponse NFκB-luc2/CD27 Jurkat cells were cultured in the presence ofhuman FcyRllb-expressing cells that can facilitate FcgR-mediatedcrosslinking of membrane-bound antibodies.

Thaw-and-Use effector FcyRIIb CHO-K1 cells (Promega, Cat # JA2251) wereplated in 96-well flat bottom culture plates (PerkinElmer, Cat # 0815),undiluted or at three increasing dilutions (⅓, ⅑. 1/27) and incubatedovernight at 37° C. / 5% CO₂. Supernatants of the adherentFcyRllb-expressing cells was replaced by a Thaw-and-Use NFκB-luc2/CD27Jurkat cell suspension of a fixed cell concentration in Bio-GloLuciferase Assay Buffer (starting at a NFκB-luc2/CD27 Jurkat : FcyRIIbCHO-K1 ratio of 1:1 for undiluted FcyRllb CHO-K1 cells), containingserial dilutions of antibody (final concentration range 0.0002 - 10µg/mL). After 6 h incubation at 37° C. / 5% CO₂, plates wereequilibrated to RT and bioluminescence was measured and presented as RLUas described in Example 2.

IgG1-CD27-A-P329R-E345R induced dose-dependent CD27 activation, whichwas independent of FcyRllb-expressing cells (FIG. 9A). In contrast, thecorresponding WT antibody IgG1-CD27-A, without the E345Rhexamerization-enhancing mutation and the P329R mutation, only showedCD27 agonism in the presence of FcyRllb-expressing cells (FIGS. 9A-E).Similarly, CD27 activation by the prior art antibodies IgG1-CD27-131A,IgG1-CD27-CDX1127 and IgG1-CD27-BMS986215 was also dependent on thepresence of FcyRllb-expressing cells and decreased gradually withdecreasing NFκB-luc2/CD27 Jurkat : FcyRllb CHO-K1 ratios(FIGS. 9 F-J).

In conclusion, these data indicate that IgG1-CD27-A-P329R-E345R caninduce CD27 agonism independent of secondary FcyR-mediatedcross-linking. This is in contrast to prior art anti-CD27 antibodiesthat were dependent on the presence of FcyR-bearing cells to induce CD27agonism.

*For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants carrying a F405Lmutation, that is functionally irrelevant in the context of thisexperiment, were used.

Example 12: Pharmacokinetic (PK) Analysis of Anti-CD27 AntibodyIgG1-CD27-A-P329R-E345R in Absence of Target Binding, Studied in Mice

The pharmacokinetic characteristics of anti-CD27 antibodyIgG1-CD27-A-P329R-E345R*, in absence of target binding, was analyzed inmice and compared to the corresponding WT antibody IgG1-CD27-A*.IgG1-CD27-A does not bind to mouse CD27 (Example 3, Table 4), and thusthe experiment was designed to test pharmacokinetic behaviour ofIgG1-CD27-A and IgG1-CD27-A-P329R-E345R in vivo, in absence of targetbinding. The study was carried out by Crown Bioscience (China) byqualified personnel, in accordance with the approved IACUC protocol andCrown Bioscience, Inc. Standard Operating Procedures. 11-12 weeks oldfemale SCID mice (C.B-17, Vital River Laboratory Animal Technology Co.,Ltd. (VR, Beijing, China; 3 mice per group) were injected intravenouslywith 500 µg antibody (25 mg/kg) in a 200 µL injection volume. 40 µLblood samples were collected at 10 minutes, 4 hours, 1 day, 2 days, 7days, 14 days and 21 days after antibody administration, plasma wascollected from blood samples and stored at -80° C. until determinationof total human IgG concentrations by ELISA. 96-well ELISA plates(Greiner, Cat # 655092) were coated overnight at 4° C. with 2 µg/mLanti-human IgG (Sanquin, The Netherlands, Article # M9105, Lot#8000260395) and subsequently blocked for 1h with PBSA (PBS supplementedwith 0.2% bovine serum albumin [BSA, Roche, Cat # 10735086001]). Next,with washing steps in between, the anti-human IgG-coated plates weresequentially incubated on a plate shaker for 1h at RT with the plasmasamples that were serially diluted in ELISA Buffer (PBSA supplementedwith 0.05% Tween 20 [Sigma-Aldrich, Cat # P1379]), for 1h at RT withpolyclonal peroxidase-conjugated goat anti-human IgG secondary antibody(Jackson, Cat # 109-035-098), and finally with2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Cat# 11112422001). The reaction was stopped by adding 2% Oxalic Acid(Riedel de Haen, Cat # 33506). Dilution series of the respectivematerials used for injection were used to generate reference curves.Absorbance was measured in an EL808 Microtiter plate reader (BioSPX) at405 nm and total human IgG concentrations (in µg/mL) were plotted.

There was no substantial difference between the PK profile ofIgG1-CD27-A-P329R-E345R and the counterpart WT antibody IgG1-CD27-A(FIG. 10 ), as determined by measuring plasma IgG levels at differenttimepoints after intravenous injection in mice.

Although a steeper decline in the initial (distribution) phase wasobserved for IgG1-CD27-A-P329R-E345R and its WT counterpart(IgG1-CD27-A) compared to predictions for human IgG1 in mice, theterminal elimination of both antibodies was in line with predictionsrates for human wild-type IgG1 based on a 2-compartment model (BleekerWK, Teeling JL, Hack CE. Blood. 2001 Nov 15;98(10):3136-42).

Together, this demonstrates that introduction of the P329R and E345Rmutations did not affect the pharmacokinetics properties of IgG1-CD27-Ain absence of target binding.

N.B. the experiment described in this example used variants ofIgG1-CD27-A and IgG1-CD27-A-P329RE345R carrying a F405L mutation, whichis functionally irrelevant in the context of this experiment.

Example 13: Induction of Antibody-Dependent Cellular Phagocytosis byAnti-CD27 Antibody IgG1-CD27-A-P329R-E345R

Antibody-dependent cellular cytotoxicity (ADCC) is mediated primarilythrough FcyRllla expressed on NK cells, whereas antibody-dependentcellular phagocytosis (ADCP) can be mediated by monocytes, macrophages,neutrophils, and dendritic cells via FcγRI, FcyRlla, and FcγRIII (Hayes,J. M et al 2016). To understand the effect of residual binding ofanti-CD27 antibody IgG1-CD27-A-P329R-E345R to FcyRla (Example 9) oneffector functions of FcyRla-expressing immune cells, the capacity ofIgG1-CD27-A-P329RE345R to induce ADCP was analyzed in vitro usingCTV-labeled CD27⁺ Burkitt’s lymphoma Daudi cells as target cells, andhuman monocyte-derived macrophages (hMDM) as effector cells (E:T = 2:1).

hMDMs were isolated from PBMCs by positive selection using CD14MicroBeads (Miltenyi Biotec, cat. no. 130-050-201), according to themanufacturer’s instruction. PBMCs were centrifuged (1,200 RPM, 5 min,RT) and resuspended in ice-cold monocyte isolation buffer (PBS, 0.5%BSA, 2 mM EDTA) at a density of 1.25 × 10⁷ PBMCs/mL. 20 µL CD14MicroBeads were added per 80 µL of PBMC suspension and incubated withagitation at 4° C. for 15 min on a rollerbank. 30 mL of ice-coldmonocyte isolation buffer was added, PBMC/CD14 MicroBeads mixturescentrifuged (300×g, 10 min, 4° C.) and resuspended in 6 mL ice-coldmonocyte isolation buffer. LS columns (Miltenyi Biotec, cat. no.130-042-401) were rinsed with 3 mL ice-cold monocyte isolation bufferand each column loaded with 3 mL PBMC/CD14 MicroBeads mixtures. Afterflow through of the CD14⁻ cells and three washes of the column inice-cold monocyte isolation buffer, CD14⁺ monocytes were recovered in 3mL of ice-cold monocyte isolation buffer by using a plunger. The CD14⁺cells were counted on a Cellometer Auto 2000 Cell Viability Counter(Nexcelom Bioscience) using ViaStain™ Viability Dye acridineorange/propidium iodide (AOPI; Nexcelom Bioscience, cat. no. CS2-0106),and resuspended at a density of 0.8 × 10⁶ cells/mL in Celgene® GMP DCmedium (CellGenix, cat. no. 20801-0500) supplemented with macrophagecolony-stimulating factor (M-CSF; Gibco, cat. no. PH9501; 50 ng/mL finalconcentration) and 3 mL of monocyte suspension (i.e., 2.4 × 10⁶monocytes) in 100 mm² Nunc™ dishes with UpCell™ Surface, which allowscell harvesting by leaving plates at RT (Thermo Fisher Scientific, cat.no. 174902). After three days of incubation, 2 mL of fresh mediumcontaining 5×M-CSF was added to the plates. After incubation for sevendays (37° C., 5% CO₂), macrophages were detached from the surface byleaving plates at RT for 1 to 1.5 h. Detached macrophages were pelletedby centrifugation, counted using AOPI, and resuspended at a density of 1× 10⁶ cells/mL in culture medium (RPMI 1640 with 10% DBSI).

Human Burkitt’s lymphoma Daudi cells (ATCC® CCL-213™) were labeled usingthe CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific,cat. no. C34557), according to the manufacturer’s instructions. Briefly,Cell Trace Violet (CTV) was added to a final concentration of 0.2 µM to1 × 10⁶ Daudi cells/mL in PBS and incubated in the dark at 37° C. for 20min (15 mL incubation volume). 10 mL DBSI was added to inactivateunbound dye. Cells were pelleted by centrifugation (300×g, 5 min),washed in PBS, and counted with AOPI. CTV-labeled Daudi cells wereresuspended at a density of 0.5 × 10⁶ cells/mL in culture medium.

For the ADCP assay, hMDM (50,000 cells/well) and CTV-labeled Daudi cells(25,000 cells/well) were seeded together (E:T = 2:1) on ice in 96-wellplates in a final volume of 150 µL culture medium and incubated withanti-CD27 antibody IgG1-CD27-A-P329R-E345R or anti-CD20 antibodyIgG1-CD20 (0.000001 to 10 µg/mL concentration range in 10-folddilutions), for 4 h (37° C., 5% CO₂). After incubation, 100 µL Human BDFc Block™ (BD Biosciences, cat. no. 564220; 1:100 in FACS buffer) wasadded and incubated at 4° C. for 10 min. Cells were pelleted bycentrifugation (300×g, 5 min), resuspended in FACS buffer containingPE-Cy7 conjugated antihuman CD11b antibody (BioLegend, cat. no. 301322;1:80) and TO-PRO-3 (Thermo Fisher Scientific, cat. no. T3605; 1:25,000)and incubated at 4° C. for 30 min. Cells were washed, resuspended inFACS buffer and collected and analyzed on a FACSymphony™ A3 CellAnalyzer (BD Biosciences). Data were analyzed using FlowJo software tomeasure viable target cell numbers and phagocytic hMDM and processed andvisualized using GraphPad Prism software.

The percentage of viable Daudi cells for each condition was calculatedaccording to the following formula:

$\begin{array}{l}{\%\mspace{6mu}\text{viable}\mspace{6mu}\text{Daudi}\mspace{6mu}\text{cells}\mspace{6mu} =} \\{\left( \frac{\%\mspace{6mu} TO - PRO - 3^{-}CD11b^{-}CTV^{+}cells\mspace{6mu} incubated\mspace{6mu} with\mspace{6mu} test\mspace{6mu} antibody}{\%\mspace{6mu} TO - PRO - 3^{-}CD11b^{-}CTV^{+}cells\mspace{6mu} incubated\mspace{6mu} without\mspace{6mu} test\mspace{6mu} antibody} \right) \times \mspace{6mu} 100}\end{array}$

The quantity of phagocytic hMDM for each condition was determined as %TO-PRO-3⁻CD11b⁺CTV⁺ cells.

IgG1-CD27-A-P329R-E345R did not increase the percentage of phagocytichMDM or reduce the percentage of viable Daudi cells in the phagocytosisassay, using hMDM from four different human healthy donors. Thisdemonstrates that residual FcyRla binding did not result inFcyRla-mediated effector functions for IgG1-CD27-A-P329R-E345R (datafrom representative human healthy donor shown in FIG. 11 ). The positivecontrol antibody IgG1-CD20 efficiently induced phagocytosis of Daudicells, that express high levels of CD20, as demonstrated by an increasein the percentage of phagocytic hMDM and a decrease in the percentage ofviable Daudi cells.

In conclusion, residual binding to FcyRla was not sufficient to induceIgG1-CD27-A-P329R-E345R-dependent ADCP of CD27⁺ cells.

Example 14: Fluid-Phase, Target-Independent, Complement Activation byAnti-CD27 Antibody IgG1-CD27-A-P329R-E345R as Determined by Measurementof C4d Deposition

Fc-Fc interaction-enhanced antibodies generally exist as monomeric IgG1molecules in solution, and hexamerize on the cell surface upon targetbinding to form a C1q docking place in case of an active Fc region(Diebolder, C. A et al 2014; de Jong, R. N et al, 2016). The IgG Fcdomain of anti-CD27 antibody IgG1-CD27-A-P329R-E345R is silenced byintroduction of the P329R mutation, which results in lack of C1q bindingto membrane-bound IgG1-CD27-A-P329R-E345R (FIG. 6 ). To confirm thatIgG1-CD27-A-P329RE345R is unable to activate complement in solution inthe absence of target binding, fluid phase, target-independent,complement activation was investigated by determination of C4ddeposition, which is considered a measure for activation of theclassical complement pathway. Fluid phase C4d fragment deposition byIgG1-CD27-A-P329R-E345R was analyzed by an enzyme-linked immunosorbentassay (ELISA) using the MicroVue™ C4d Enzyme Immunoassay (EIA; Quidel,cat. no. A008) and was performed according to the manufacturer’sprotocol. Heat Aggregated Gamma Globulin (HAGG; Complement Activator;Quidel, cat. no. A114) was used as a positive control for the assay.IgG1-b12 and IgG1-b12-RGY (WO2014006217A1)) were included as controlantibodies. Introduction of E345R/E430G/S440Y (RGY) Fc mutations in anIgG1 antibody has been described to induce the formation of hexamers insolution, resulting in fluid phase complement activation (Diebolder, C.A et al, 2014; Wang, G., R. N et al, 2016; de Jong, R. N et al , 2016).IgG1-b12-P329R-E345R was included as isotype control antibody.

Antibody dilutions were prepared in phosphate-buffered saline (PBS) to aconcentration of 1 mg/mL, except for HAGG, which was diluted to aconcentration of 10 mg/mL. Then, the test samples were further dilutedto a concentration of 100 µg/mL (for monoclonal IgG) or 1,000 µg/mL (forHAGG) in 90% (final concentration) normal human serum (NHS) (CompTech,Lot. no. 42a) and incubated at 37° C. for 1 h. In parallel, ‘Noantibody’ samples (no antibody, 90% NHS) and ‘PBS only’ samples (noantibody, no NHS) were included as negative controls. Next, the sampleswere diluted 1:250 in cold kit-provided Complement Specimen Diluent. Inthe meantime, the strips coated with mouse anti-human C4d antibody wereplaced in a 96-wells plate and the assay wells were washed three timeswith 250 to 300 µL Wash Buffer with a 1-min waiting step after the firstwash. The test samples were added to the wells (100 µL/well) and as anegative control, Complement Specimen Diluent only (blank) was used inthe ELISA. In parallel, 100 µL of the standards (Standard A-E) andinternal controls provided by the kit were added to separate wells. Theplates were incubated for 30 min at RT. Then, the plates were washedfive times with Wash Buffer as described above. 50 µL of C4d Conjugate(peroxidase-conjugated goat anti-human C4d) was added to the wells andthe plates were incubated for 30 min at RT. After five washing stepswith Wash Buffer as described above, 100 µL of C4d Substrate [0.7%2-2′-Azino-di-(3-ethylbenzthiazoline sulfonic acid diammonium salt] wasadded and again the plates were incubated for 30 min at RT. Finally, 50µL kit-provided Stop Solution was added and within 1 h, the opticaldensity was measured at 405 nm using an ELISA Plate Reader (EL808BioSPX, BioTek).

IgG1-CD27-A-P329R-E345R and the control antibody IgG1-b12-P329R-E345R(having the same Fc backbone as IgG1-CD27-A-P329R-E345R) did not inducefluid phase C4d deposition at the tested concentration of 100 µg/mL; themeasured C4d levels were similar to background levels of the controlantibody with a wild-type Fc domain (IgG1-b12) and the no antibodycontrol (FIG. 12 ). In contrast, the positive control antibodyIgG1-b12-RGY, that is known to form hexamers in solution, induced C4ddeposition to the same level as HAGG.

These data show that IgG1-CD27-A-P329R-E345R did not inducetarget-independent, fluid phase complement activation in vitro.

Example 15: Capacity of Anti-CD27 Antibody IgG1-CD27-A-P329R-E345R toCompete for Ligand-Binding With CD70

To determine if anti-CD27 antibody IgG1-CD27-A-P329R-E345R interfereswith the interaction of CD27 with its natural ligand CD70, binding of asaturating concentration of biotinylated recombinant human CD70extracellular domain (ECD) to CD27, endogenously expressed on humanBurkitt’s lymphoma cell line Daudi, was studied in the presence andabsence of excess IgG1-CD27-A-P329R-E345R.

Daudi cells (ATCC® CCL-213™) cultured in RPMI 1640 medium (Gibco, cat.no. A10491-01) supplemented with 10% donor bovine serum with iron (DBSI;Gibco, cat. no. 20731-030) were seeded at 50,000 cells/well in roundbottom 96-well plates (Greiner Bio One, cat. no. 650261). Cells werepelleted by centrifugation (300×g, 3 min at 4° C.) and resuspended inFACS buffer (PBS, 1% BSA [Roche, cat. no. 1073508600]) containinganti-CD27 or control antibodies (50 µg/mL final concentration).Biotinylated recombinant human CD70 ECD (Abcam, cat. no. ab271443) wasadded at a saturating concentration (6 µg/mL) and cells were incubatedat 4° C. for 30 min.

Cells were washed twice and resuspended in FACS buffer containingBrilliant Violet (BV) 421™ labeled streptavidin (BioLegend, cat. no.405225; 0.0025 µg/mL final concentration) and R phycoerythrin (PE)labeled polyclonal AffiniPure F(ab′)₂ fragment goat-anti-human IgG Fc(Jackson ImmunoResearch, cat. no. 109 116098; 0.0025 µg/mL finalconcentration) at 4° C. for 30 min. Cells were washed twice, resuspendedin FACS buffer containing TO-PRO-3 iodide (Thermo Fisher Scientific,cat. no. T3605; 1:25,000) and analyzed. Data were collected on a BDFACSymphony™ A3 flow cytometer (BD Biosciences) and analyzed usingFlowJo software. For compensation, one drop of UltraComp eBeads™Compensation Beads (Life Technologies, cat. no. 01-2222-42) was added toeach well. 2 µL of each antibody was added and mix was incubated for 20min. Plates were spun down and beads were resuspended in FACS buffer andmeasured. For viability compensation, cells were treated at 65° C. for10 min and mixed 1:1 with viable cells. Cells were spun down andresuspended in TO-PRO-3 diluted in FACS buffer. Data were processed andvisualized using GraphPad Prism.

IgG1-CD27-A-P329R-E345R or IgG1-CD27-A did not block binding of the CD70ECD to CD27⁺ Daudi cells, as CD70 binding levels were comparable tothose for Daudi cells incubated with the nonbinding isotype controlantibodies IgG1-b12-P329R-E345R or IgG1-b12, or cells without antibody(FIG. 13 ). Also, prior art anti-CD27 antibodies IgG1-CD27-BMS986215 andIgG1-CD27-131A showed a weak blocking effect on CD27 binding to CD70ECD. In contrast, CD70 was unable to bind to surface CD27 on Daudi cellsin presence of prior art anti-CD27 antibody IgG1-CD27-CDX1127 (FIG. 13 )that was previously reported to block ligand-binding (Vitale et al,2012).

In conclusion, IgG1-CD27-A-P329R-E345R binding does not block CD27binding by its natural ligand CD70 on Daudi cells.

Example 16: T-Cell Activation Marker Expression Upon Incubation ofPolyclonally Stimulated Human PBMC with Anti-CD27 Antibodies

The effect of IgG1-CD27-A-P329R-E345R on expression of T-cell activationmarkers in polyclonally activated T cells was studied using PBMCobtained from three different healthy human donors. Expression ofHLA-DR, CD25, CD107a, and 4-1BB were analyzed after incubating PBMCswith IgG1-CD27-A-P329RE345R or prior art anti-CD27 antibodies for twoand five days.

Freshly isolated 75,000 PBMC/well were seeded in 96-well U bottom plates(Greiner Bio-One) in cell culture medium. Duplicate wells were incubatedsimultaneously with anti-CD3 antibody (UCHT1 clone; Stemcell; 0.1µg/mL); and IgG1-CD27-A-P329R-E345R (0.0005 to 30 µg/mL in threefolddilutions); or prior art anti-CD27 antibodies IgG1-CD27-CDX1127,IgG1-CD27-131A, and IgG1-CD27-BMS986215 (30 µg/mL); or nonbindingcontrol antibody IgG1-b12-P329R-E345R (10 µg/mL). To determineexpression of each activation marker in absence of treatment, duplicatecontrol wells with untreated (no anti-CD3 or anti-CD27 antibodies) cellswere supplemented with culture medium alone. To set the gates foridentifying activation marker positive cells, fluorescence minus one(FMO) controls were used. For the FMO controls, all the antibodies usedin the experiment except for one corresponding to an activation markerin duplicate wells was added to 75,000 PBMC/well from one donoractivated with anti-CD3 antibody. Untreated cells from each donor insingle wells with no staining antibody were included as negativecontrols. To detect viable cells, untreated cells from each donor werestained with 4′,6-diamidino-2-phenylindole (DAPI) alone in single wells.

After incubation for two or five days (37° C., 5% CO₂), plates werewashed once with FACS buffer and resuspended in an antibody mixture inFACS buffer containing antibodies for T-cell activation markers 4-1BB,CD25, CD107a, human leukocyte antigen (HLA)-DR; and antibodies forgating CD4⁺ and CD8⁺ T-cell subsets in flow cytometry. After incubationat 4° C. for 30 min, all plates were washed twice with FACS buffer andcells were resuspended in FACS buffer. The samples were analyzed on a BDLSRFortessa Cell Analyzer using FlowJo software to determine the medianfluorescence intensity (MFI) and percentage of positive cells for eachT-cell activation marker on CD4⁺ and CD8⁺ T cells. Anti-CD27 antibodyinduced changes in the expression levels of the T-cell activationmarkers were presented as the fold change in MFI of the anti-CD27antibody sample relative to the nonbinding control antibodyIgG1-b12-P329R-E345R. The samples were analyzed on a BD LSRFortessa™Cell Analyzer (BD Biosciences) using FlowJo software.

IgG1-CD27-A-P329R-E345R increased expression of CD25, CD107a and 4-1BBon activated CD4⁺ T cells (FIG. 14A). These effects were more pronouncedafter 2 days of incubation than after 5 days of incubation. On CD8⁺ Tcells, incubation with IgG1-CD27-A-P329R-E345R resulted in an increasedexpression of HLA-DR, CD107a and 4-1BB both after 2 and 5 days ofincubation (FIG. 14B).

The expression of T-cell activation markers was also assessed uponincubation for 2 and 5 days with three prior art antibodies.IgG1-CD27-131A and IgG1-CD27-BMS986215 induced a comparable increase inexpression of HLA-DR, 4-1BB, CD25, and CD107a on CD4⁺ and CD8⁺ T cells,while the effect of incubation for 2 or 5 days with IgG1-CD27-CDX1127 onT-cell activation marker expression was less pronounced.

In conclusion, incubation of polyclonally activated PBMC withIgG1-CD27-A-P329R-E345R resulted in an increased expression ofactivation markers HLA-DR, CD25, CD107a and 4-1BB on CD4⁺and CD8⁺Tcells.

Example 17: Percentages of OVA-Specific CD8⁺ T Cells in OVAProtein-Immunized Mice After Injection of Anti-CD27 Antibodies in aHuman CD27-KI Mouse Model

The effect of IgG1-CD27-A-P329R-E345R treatment on expansion ofantigen-specific T cells in the hCD27 KI OVA model in splenocytes wasanalyzed by flow cytometry.

Homozygous human CD27 (hCD27)-KI mice on a C57BL/6 background (hCD27 KImice) were obtained from Beijing Biocytogen Co., Ltd. (strain nameC57BL/6-Cd27tm1(CD27)/Bcgen, Stock no. 110006). This strain wasdeveloped in collaboration with the HuGEMM™ platform of CrownBioscience, featuring a humanized drug target (CD27 in this case) withinmice with a functional immune system. In hCD27 KI mice, exons 1-5 of themouse CD27 gene encoding the extracellular domain were replaced by humanCD27 exons 1-5. OVA-specific T cells were induced in vivo bysubcutaneous (s.c.) injection of the immunogen ovalbumin (OVA) inhCD27-KI mice and the agonist effect of IgG1-CD27-A-P329R-E345R wastested by simultaneously treating the mice intravenously (i.v.) with theantibody.

On day 0, the mice were injected s.c. with 5 mg OVA (InvivoGen, cat. no.vac-pova-100, lot. no. EFP-42-04) and treated by i.v. injection into thetail vain with IgG1-CD27-A-P329R-E345R (30 mg/kg), IgG1-CD27-CDX1127 (30mg/kg) or IgG1-b12-P329R-E345R (30 mg/kg). On day 12 and day 21, micewere boosted with OVA and treated with antibody as on day 0. On day 10,day 19 and day 24, blood was collected via cheek pouch or saphena in BDMicrotainer® blood collection tubes containing di-potassiumethylenediaminetetraacetic acid (K2-EDTA; BD, cat. no. 365974) andimmediately used in further analysis. On day 28, mice were euthanizedand spleens were resected under sterile conditions.

Resected spleen tissue in RPMI1640 medium (Thermo Fisher Scientific,cat. no. C22400500BT) was transferred to gentleMACs™ CTubes (MiltenyiBiotec, cat. no. 130-093-237) and mechanically dissociated to a singlecell suspension using the gentleMACS™ Dissociator (Miltenyi, cat. no.130-093-235), according to the manufacturer’s instructions. Afterdissociation, the cell suspension was filtered through a 70 µm cellstrainer (Falcon, cat. no. 352350). Next, samples were washed twice byresuspension in 3 mL wash buffer (sterile PBS [Hyclone, SH0256.01B]supplemented with 4% FBS [Gibco, cat. no. 10099 141]). Cells werecounted on a Cellometer Auto T4 (Nexcelom Bioscience) and the number ofcells was adjusted to 2 × 10⁶ splenocytes per tube.

2 × 10⁶ splenocytes were transferred to FACS tubes (Falcon, cat. no.352052) and resuspended in wash buffer (sterile PBS [Hyclone,SH0256.01B] supplemented with 4% FBS [Gibco, cat. no. 10099 141])supplemented with 1 µg/mL purified rat anti-mouse CD16/CD32 (Mouse BD FcBlock™, BD Biosciences, cat. no. 553141). After a preincubation at 2-8°C. for 10 min in the dark, 10 µL PE-labeled OVA-tetramer (MBL Lifescience, cat. no. TS 5001 1C) was added, and the samples were gentlyvortexed before further incubating at 2-8° C. for 30-60 min in the dark.Without washing, labeled antibodies and compounds used for flowcytometry gating of T-cell subsets were added. The samples were gentlyvortexed and incubated at 2-8° C. for an additional 30 min in the dark.Next, samples were washed twice by resuspension in 2 mL wash buffer andcentrifuged at 300×g for 5 min. Finally, the cells were resuspended in250 µL wash buffer and analyzed on a BD LSRFortessa™ X-20 Cell Analyzer(BD Biosciences). Data were processed using Kaluza Analysis Software(Beckman Coulter).

IgG1-CD27-A-P329R-E345R increased the percentages of OVA-specific CD8⁺Tcells in the blood and spleen of mice simultaneously injected with OVAprotein vaccination. The percentages of OVA-specific CD8⁺ T cells inmice treated with 30 mg/kg IgG1-CD27-CDX1127 were lower than theIgG1-CD27-A-P329R-E345R-treated group and comparable to theIgG1-b12-P329R-E345R-treated group (FIG. 15 ).

Example 18: IFNy Secretion by OVA-Specific CD8⁺ T Cells from spleens ofOVA-Immunized Mice Injected With Anti-CD27 Antibodies

Resected spleen tissue in RPMI1640 medium (see Example 17) was gentlymashed over a 70 µm cell strainer (Falcon, cat. no. 352350), pelleted bycentrifugation (1,500 rpm, 5 min), and resuspended in 10 mLAmmonium-Chloride-Potassium (ACK) Lysing Buffer (Invitrogen, cat. no.A1049201). After 3-5 min incubation at RT, samples were washed twicewith 10-20 mL PBS and resuspended in 5 mL Cellular Technology Limited(CTL) Test™ Medium (ImmunoSpot, cat. no. CTLT-005) supplemented with 50U/mL penicillin and 50 µg/mL streptomycin (pen/strep, Gibco, cat. no.15070-063). The collected splenocytes were filtered again through a 70µm cell strainer and counted on a Vi-CELL™ XR Cell Viability Analyzer(Beckman Coulter) to adjust the concentration to 3.125 × 10⁶ cells/mLwith CTL-Test Medium containing pen/strep.

IFNy production by splenocytes was analyzed using the Mouse IFN-yELISpotPLUS kit (Mabtech, cat. no. 3321-4HPW-2), essentially asdescribed by the manufacturer. Pre-coated MultiScreenHTS IP Filter(MSIP) white plates (mAb AN18) were washed four times with 200 µLsterile PBS per well and conditioned with 200 µL CTL-Test Mediumcontaining pen/strep (RT, 30 min). Medium was removed and 5 × 10⁵splenocytes/well were incubated in duplicate with 2 µg/mL OVA₂₅₇₋₂₆₄peptide SIINFEKL (Invivogen, cat. no. vac-sin), or scrambled controlpeptide FILKSINE (SB-PEPTIDE, cat. no. SB073-1MG) in a total volume of180 µL/well for 20 h in a humidified incubator (37° C., 5% CO₂). As apositive control for IFNy production, splenocytes were incubated inparallel with a cell stimulation cocktail consisting of 500 ng/mLphorbol myristate acetate (PMA) and 10 µg/mL ionomycin (PMA+lonomycin,Dakewe Biotech, cat. no. DKW ST PI). Cultures of splenocytes withoutpeptide were included as a negative control. After incubation, the cellswere removed and the plates were washed five times with PBS. Next,plates were sequentially incubated, with five wash steps with PBS inbetween, with Biotinylated detection mAb (R4-6A2;RT,2h),Streptavidin-horseradish peroxidase (HRP; RT, 1 h), and finally3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (all provided bythe kit). When distinct spots emerged, the reaction was stopped bywashing extensively in deionized water. Spots were counted on an AIDiSpot ELISpot Reader (Autoimmun Diagnostika [AID] GMBH, ELR08IFL) usingspotAID V8 software (AID). ELISpot data were analyzed and presented inbar diagrams using GraphPad Prism software and presented as the meannumber of spots per well ± SEM from all mice per treatment group (n=5).

Splenocytes from all IgG1-CD27-A-P329R-E345R-treated animal groupsshowed increased IFNy production in response to treatment with OVApeptide, as demonstrated by ELISpot analysis (FIG. 16 ). Stimulation ofthe splenocytes with a scrambled control peptide induced no or minimalIFNy production, suggesting that IFNy was produced by OVA-specific Tcells. In contrast, no IFNy production was observed in splenocytes frommice treated with 30 mg/kg IgG1-CD27-CDX1127.

Example 19: Effect of IgG1-CD27-A-P329R-E345R Treatment on T-CellActivation in OVA-Immunized Mice in Vivo

The effect of IgG1-CD27-A-P329R-E345R treatment on CD8⁺ T-cellactivation was studied in vivo by analyzing the expression of PD-1 onCD8⁺ T cells derived from OVA-treated hCD27-KI mice. Mice were treatedas described in Example 17. Also, methods to obtain and analyzesplenocytes by FACS are described in Example 17.

IgG1-CD27-A-P329R-E345R induced an increase in the percentage of CD8⁺ Tcells expressing activation marker PD-1 on day 28. CD8⁺PD-1⁺ T-cellpercentages were low in animals treated with IgG1-CD27-CDX1127 orcontrol antibody IgG1-b12-P329R-E345R (FIG. 17 ).

Example 20: Effect of IgG1-CD27-A-P329R-E345R Treatment on in VivoInduction of T-Cell Subsets in OVA-Immunized Mice

The effect of IgG1-CD27-A-P329R-E345R on the expansion of T-cell subsetswas studied by analyzing the expression of CD44 and CD62L in splenocytesamples from OVA-treated hCD27-KI mice. Memory CD8⁺ T cells derived fromspleens of IgG1-CD27-A-P329R-E345R-treated, OVA-immunized, hCD27-KI micewere quantified by flow cytometry. Memory T cells were classified aseffector memory (CD44⁺CD62L⁻) and pre-effector T cells (CD44⁻CD62L⁻;Nakajima, Y., K et al 2018). Mice were treated as described in Example17. Also, methods to obtain and analyze splenocytes by FACS aredescribed in Example 17.

IgG1-CD27-A-P329R-E345R (30 mg/kg) induced increased percentages ofpre-effector T cells and effector memory CD8⁺T cells in the spleen onday 28 when compared to splenocytes of mice treated withIgG1-b12-P329R-E345R (FIG. 18 ). Within the CD45⁺ population,IgG1-CD27-A-P329R-E345R induced higher percentages of pre-effector Tcells and effector memory T cells than IgG1-CD27-CDX1127 (30 mg/kg),while comparable mean percentages of these T-cell populations wereinduced by both anti-CD27 antibodies in the CD8⁺ fraction ofsplenocytes.

Example 21: Effect of IgG1-CD27-A-P329R-E345R Treatment on in VivoExpansion of T Cells in OVA-Immunized Mice

The effect of IgG1-CD27-A-P329R-E345R on expansion of T cells wasstudied by analyzing the expression of CD3 in splenocyte and bloodsamples from OVA-treated hCD27-KI mice. Mice were treated as describedin Example 17. Also, methods to obtain and analyze splenocytes and bloodsamples by flow cytometry are described in Example 17.

Treatment of OVA-immunized hCD27-KI mice with 30 mg/kgIgG1-CD27-A-P329R-E345R did not increase the percentage of CD3⁺T cellsin the spleen, compared to treatment with the non-binding controlantibody IgG1-b12-P329R-E345R (FIG. 19 ). In contrast, treatment withbenchmark antibody IgG1-CD27-CDX1127 (30 mg/kg) resulted in a decreaseof CD3⁺ T cells in the spleen. Similar observations were made inperipheral blood samples.

Example 22: Effect of IgG1-CD27-A-P329R-E345R on T-Cell CytokineProduction in Antigen-Specific Studies

The capacity of IgG1-CD27-A-P329R-E345R to increase cytokine productionwas studied using T cells that had been stimulated by their cognateantigen. PBMC were isolated from buffy coats obtained from healthy humandonors by Ficoll-Paque density gradient separation (GE Healthcare, cat.no. 17 1440 03) according to the manufacturer’s instructions.

Human magnetic CD14 and CD8 MicroBeads (Miltenyi Biotec, cat. no. 130050 201 and 130 045 201, respectively) were used for positive selectionof CD14⁺ monocytes and negative selection of CD14⁻ PBL from human PBMC,and positive selection of CD8⁺ T cells from frozen PBL. Cell suspensionswere centrifuged and resuspended in magnetic-activated cell sorting(MACS) buffer (Dulbecco’s phosphate-buffered saline [DPBS] with 5 µMEDTA and 0.2% human albumin) at 1 × 10⁷ live cells per 80 µL MACSbuffer. Per 1 × 10⁷ cells, 12 µL CD14 or CD8 MicroBeads were added.Subsequent MACS separation was performed using an automated magneticcell separation instrument or by manual separation. Automated MACSseparation was performed using an autoMACS® Pro Separator (MiltenyiBiotec), according to the manufacturer’s instructions. Eluted CD14⁺monocytes and CD8⁺ T cells were centrifuged (8 min, 300×g at RT)resuspended in X-VIVO 15 medium (Lonza), and counted with erythrosine Bsolution for further use; i.e., monocyte differentiation into iDC orelectroporation of CD8⁺ T cells with PD-1 and/or CLDN6-specific T-cellreceptor (TCR) mRNA.

For the generation of monocyte-derived iDC, up to 40 × 10⁶ PBMC-derivedCD14⁺ monocytes were cultured (37° C., 5% CO₂) for five days in T175flasks in DC medium (RPMI 1640, 5% pooled human serum [PHS; One Lambda,cat. no. A25761], 1× minimum essential medium non-essential amino acidsolution [MEM NEAA, Life Technologies, cat. no. 11140 035], 1 mM sodiumpyruvate [Life Technologies, cat. no. 11360 039]) supplemented with 100ng/mL human granulocyte/macrophage colony-stimulating factor (GM-CSF;Miltenyi Biotec, cat. no. 130-093-868) and 50 ng/mL human IL-4 (MiltenyiBiotec, cat. no. 130093 924). After three days in culture, half of themedium per flask was replaced. Nonadherent monocytes in the medium takenfrom the flask were pelleted (8 min, 300×g at RT), resuspended in freshDC medium supplemented with 200 ng/mL GM-CSF and 100 ng/mL IL-4, andthen returned into the originator flask. After the five days ofincubation, the iDC which adhered to the culture flask were detachedusing 10 mL DPBS containing 2 mM EDTA (37° C., 10 min). The isolated iDCwere washed, pelleted (8 min, 300×g at RT) and used for electroporationwith CLDN6 mRNA.

Human CD8⁺ T cells were electroporated with RNA encoding the alpha andbeta chains of a mouse TCR specific for human CLDN6, either alone ortogether with RNA encoding PD-1, and human monocyte-derived iDC wereelectroporated with RNA encoding human CLDN6. Up to 5 × 10⁶ iDC or 15 ×10⁶ CD8⁺ T cells were electroporated in 250 µL X-VIVO 15 medium at RTusing an ECM 830 Square Wave Electroporation System (BTX®). Cells weremixed with RNA, pulsed (500 V, 3 ms for T cells or 300 V, 12 ms foriDC), and immediately diluted with 750 µL pre-warmed assay medium (IMDMGlutaMAX [Life technologies, cat. no. 31980030] with 5% PHS).Electroporated iDC were transferred to 6- or 12-well plates and culturedO/N (37° C., 5% CO₂). After O/N incubation, electroporated CD8⁺ T cellsand iDC were evaluated by flow cytometry to evaluate cell purity,expression of transfected RNA (PD-1 and CLDN6-TCR on CD8⁺ T cells andCLDN6 on iDC), and baseline expression of CD27 and PD-1 on CD8⁺ T cellsand PD-L1 on iDC. Approximately 78% to 93%, 78% to 92%, and 36% to 98%of electroporated CD8⁺T cells expressed CLDN6-TCR, PD-1, and endogenousCD27, respectively. Approximately 47% to 91% and 94% to 99% ofelectroporated iDC expressed CLDN6 and endogenous PD-L1, respectively(not shown).

CD8⁺ T cells and iDC were seeded at a 10:1 ratio (7.5×10⁴ T cells and7.5×10³ iDC per well) in a 96-well round-bottom plate.IgG1-CD27-A-P329R-E345R was diluted in assay medium and 25 µL of dilutedIgG1-CD27-A-P329R-E345R was added to the wells, to reach a finalconcentration of 10 µg/mL. Similarly, the control antibodiesIgG1-CD27-131A and IgG1-b12-P329R-E345R were added to reach finalconcentrations of 10 µg/mL. Antigen-specific T-cell activity uponantibody treatment was analyzed in vitro by measuring cytokines in thesupernatant of T cells transduced to express CLDN6-TCR, which wereco-cultured with iDC transduced to express and present CLDN6.Supernatants were collected after two days, and concentrations ofmultiple proinflammatory cytokines and chemokines were determined bymultiplex electrochemiluminescence assays (ECLIA) using the 10-spotU-PLEX ImmunoOncology Group 1 (human) kit (MSD; cat. no. K151AEL 2)following the manufacturer’s instructions.

For the 10-spot U-PLEX Immuno-Oncology Group 1 kit, biotinylated captureantibodies were preincubated at RT with the assigned linkers, which havea biotin-binding domain, for 30 min, followed by 30 min incubation withStop Solution. Plates were coated with a mix of the linker coupledcapture antibodies by incubating at RT with shaking for 1 hr. Plateswere washed three times with 1× MSD Wash Buffer. Supernatant samples orkit standards were diluted 1:2 in Assay Diluent, added to the wells andincubated at RT for 2 h with constant shaking. The plates were washedthree times with Wash Buffer, and incubated with SULFO-TAG-conjugateddetection antibodies from the kit at RT for 1 h with constant shaking.The plates were washed three times with Wash Buffer before adding ReadBuffer B to catalyze the electrochemiluminescent reaction. The plateswere immediately analyzed by measuring light intensity on a MESOQuickPlex SQ 120 imager (MSD).

IgG1-CD27-A-P329R-E345R-induced changes in cytokine production wereassessed by multiplex ECLIA in supernatants from the CD8⁺ T cell/iDCco-cultures after two days of incubation (n=4 different donors).IgG1-CD27-A-P329R-E345R induced a significant increase in the productionof GM-CSF and IFN-y in CD8⁺ T cell/iDC co-cultures with CD8⁺ T cellsexpressing endogenous levels of PD-1 (FIG. 20A), while also an increasein IL-13 and TNFα production was observed. A considerable increase forthe same cytokines was observed in cultures containingPD-1-overexpressing T cells (FIG. 20B). While cytokine levels weregenerally decreased when T cells overexpressed PD-1, the relativeincrease (fold increase) in cytokine production in presence ofIgG1-CD27-A-P329R-E345R was highest in this setting (FIGS. 20A and B).In contrast, prior art anti-CD27 antibody IgG1-CD27-131A showed minimaleffect on cytokine production compared to the nonbinding controlantibody IgG1-b12-P329R-E345R (FIGS. 20A and B).

Example 23: Expression of Cytotoxicity-Associated Molecules byAntigen-Specific CD8⁺ T Cells Incubated With IgG1-CD27-A-P329R-E345R

The induction of T-cell mediated cytotoxicity upon antibody treatmentwas studied by analyzing the expression of cytotoxicity-associatedmolecules on the antigen-specific T cells by flow cytometry inco-cultures of human healthy donor T cells transduced to express aCLDN6-TCR and MDA-MB-231_hCLDN6 target cells.

MDA-MB-231_hCLDN6 cells were generated by lentiviral transduction. Tothis end, 2×10⁵ MDA-MB-231 cells in 250 µL Dulbecco’s modified eaglemedium (DMEM, Thermo Fisher Scientific, cat. no. 31966-047) supplementedwith 10% FBS (non-heat-inactivated) were seeded per well in a 12-welltissue culture plate.

The cells were incubated for 1-2 h at 37° C. (7.5% CO₂). Supernatantscontaining lentiviral vectors encoding human CLDN6(pL64b42E(EF1a-hClaudin6)Hygro-T2A-GFP) were thawed on ice and dilutedin a total volume of 750 µL DMEM/10% FBS to obtain titers of 2×10⁵,8×10⁴, and 3.2×10⁴ TU/mL. These titers corresponded to MOI of 1, 0.4,and 0.16, respectively. The supernatants were then added to theMDA-MB-231 cells, and the cells were incubated for 72 h at 37° C. (5%CO₂) without disturbance. For the experiments described in the currentExample, MDA-MB-231-hCLDN6 cells were cultured in DMEM/10% FBS. Cellswere passaged or harvested for experiments at 70% to 90% confluence.Cells were detached by treatment with Accutase (Thermo FisherScientific, cat. no. A11105010) for 5 min (37° C., 7.5% CO₂), andresuspended by addition of culture medium. Cells were centrifuged(300×g, 4 min at RT) and counted. MDA-MB-231_hCLDN6 cells were notcultured for more than 20 passages.

MDA-MB-231_hCLDN6 cells were seeded at 1.2 to 1.5 × 10⁴ cells/well, in96-well flat-bottom plates (for flow cytometry analysis) and xCELLigenceE-plates (Agilent, cat. no. 05232368001; for impedance measurement) andallowed to settle at RT for 30 min. Next, plates were incubated for oneday in the incubator and the xCELLigence real-time cell analysis (RTCA)instrument (ACEA Biosciences), respectively (37° C., 5% CO₂).

Isolated CD8⁺T cells (see Example 22) were electroporated withCLDN6-specific TCR mRNA and incubated O/N. After CD8⁺ T-cell isolationand electroporation, T-cell cultures contained 49% to 99% CD8⁺ T cells.Of these electroporated CD8⁺ T cells, approximately 78% to 93% expressedCLDN6-TCR and 59% to 98% of CLDN6-TCR⁺ CD8⁺ cells were CD27⁺. Cells werecentrifuged (8 min, 300×g at RT), resuspended in DMEM/10% FBS andcounted. The cells were centrifuged again, resuspended at 3 × 10⁶cells/mL in DMEM/10% FBS, and added to the wells containing thepreviously seeded MDA-MB-231_hCLDN6 cells (1.5 × 10⁵ CD8⁺T cells/well; Tcell:tumor cell, effector:target, ratio of 10:1).IgG1-CD27-A-P329R-E345R, IgG1-CD27-131A, and the nonbinding controlantibody IgG1-b12-P329R-E345R were added to the co-cultures at 10 µg/mL.CD107a and GzmB expression were determined by flow cytometry.

After two days of incubation in the presence of 10 µg/mLIgG1-CD27-A-P329R-E345R, the percentage of GzmB⁺CD107a⁺CD8⁺ T cells wassignificantly enhanced compared to treatment with the nonbinding controlantibody or prior art anti-CD27 antibody IgG1-CD27-131A (FIG. 21 ).

In conclusion, these data show that IgG1-CD27-A-P329R-E345R was able toinduce cytotoxicity-associated molecules on activated antigen-specific Tcells.

Example 24: Capacity of IgG1-CD27-A-P329R-E345R to Induce T-CellMediated Tumor Cytotoxicity

To evaluate T-cell mediated cytotoxicity, CLDN6-TCR-electroporated CD8⁺T cells were co-cultured with MDA-MB-231_hCLDN6 cells in the presence ofIgG1-CD27-A-P329R-E345R, prior art anti-CD27 antibody IgG1-CD27-131A, ornonbinding control antibody IgG1-b12-P329R-E345R for five days in anxCELLigence real-time cell analysis instrument (Acea Biosciences), withimpedance measurements at two-hour intervals, as described in Example23. Cell index values were derived from impedance measurements conductedat two-hour intervals. Area-under-the-curve (AUC) were obtained fromcell index data over five days of co-culture. AUC were normalized toco-cultures treated with IgG1-b12-P329R-E345R. The magnitude ofimpedance is dependent on cell number, cell morphology, and cell sizeand on the strength of cell attachment to the plate, which altogether isused in this particular case as an indirect readout of tumor cell mass.Decrease in impedance in this experimental setting is considered asurrogate of tumor-cell killing by CD8⁺ T cells. It should be noted thatimpedance may underestimate tumor cell killing due to proliferation of Tcells.

IgG1-CD27-A-P329R-E345R induced a decrease in cell index, indicative oftumor-cell killing. IgG1-CD27-131A did not have a visible effect on cellindex, indicating minimal capacity to increase tumor-cell killing (FIG.22 ).

Example 25: Capacity of IgG1-CD27-A-P329R-E345R to Induce Expansion ofTumor-Infiltrating Lymphocytes

The capacity of IgG1-CD27-A-P329R-E345R to induce expansion oftumor-infiltrating lymphocyte (TIL) subsets (CD4⁺ and CD8⁺ T cells, NKcells, and regulatory T cells [Treg]) was evaluated ex vivo usingcryopreserved tumors that had been surgically resected from NSCLCpatients.

Surgically resected human NSCLC tissues were received in transportmedium (HypoThermosol® FRS Preservation Solution [BioLife Solutions,cat. no. 101104], 7.5 µg/mL Amphotericin B [Thermo Fisher Scientific,cat. no. 15290026], and 300 units/mL (U/mL) pen/strep [Thermo FisherScientific, cat. no. 15140-122]). Samples were washed three times inwash medium (5 mL X-VIVO 15 [Lonza], 2.5 µg/mL Amphotericin B, [ThermoFisher Scientific] and 100 U/mL pen/strep [Thermo Fisher Scientific])and transferred to a cell culture dish. Fatty tissue and necrotic areaswere removed with a scalpel, and the tissue was cut into fragments ofapproximately 5 mm³. Each fragment was placed in an individual cryovial,and 1 mL freezing medium (FBS, 10% DMSO) was added to each vial. Thevials were transferred into a controlled freeze-chamber (Mr. Frostyfreezing container), which was placed in a -80° C. freezer. After atleast 16 h at -80° C., the vials were transferred to liquid nitrogen forlong-term storage.

4 to 6 cryopreserved vials containing tumor fragments of approximately 5mm³ from one tumor specimen were thawed per experiment in a 37° C. waterbath for approximately 2 min and washed five times with wash medium andtransferred to a cell culture dish. The tumor fragments were furtherdissected with a scalpel into fragments of approximately 1 mm³. Most ofthe fragments were used for TIL expansion upon culturing with IL-2 andtreatment antibody and remaining fragments were used to determineexpression of specific cell surface markers at baseline, without anytreatment.

Two tumor fragments per well (on average) were seeded in 24-well plates(2 mL/well total volume capacity used in assay) in 0.1 mL prewarmed TILcultivation medium (X-VIVO 15 [Lonza] with 2% human serum albumin [HSA;CSL Behring, cat. no. PZN-00504775], 100 U/mL pen/strep [Thermo FisherScientific], and 2.5 µg/mL Amphotericin B [Thermo Fisher Scientific])containing 45 to 50 U/mL IL-2 (Proleukin S; Novartis Pharma, cat. no.PZN-02238131). IgG1-CD27-A-P329R-E345R was diluted in TIL cultivationmedium containing 45 to 50 U/mL IL-2 and 900 µL of this dilution wasadded to the wells as appropriate. Final IgG1-CD27-A-P329R-E345Rconcentrations in the wells were 1 or 10 µg/mL. As a control, mediumcontaining 45 to 50 U/mL IL-2 without antibodies was added to tumorfragments in separate wells. A total of 8 to 16 wells were incubated foreach experimental condition per donor (37° C., 5% CO₂).

After three days of culture, fresh TIL cultivation medium containing 45to 50 U/mL IL-2 and IgG1-CD27-A-P329R-E345R was added to the wells (1mL/well, same antibody concentrations as above). Between day 5 and 14/17after assay initiation, the cultures were regularly monitored with amicroscope for proliferation of TIL that migrated from the tissuefragments and the formation of TIL microclusters. If >25 TILmicroclusters were observed in one well after seven or eight culturedays, cells and tissue fragments from two identically treated originalwells were resuspended and pooled into one well of a 6-well plate (5 to6 mL/well total volume capacity used in assay) with the culture mediumand fresh IL 2 containing TIL cultivation medium was added (estimated 33U/mL IL-2 final concentration).

Every two to three days, cultures were supplemented with freshIL-2-containing TIL cultivation medium. IL-2 concentrations in themedium added to cultures were reduced to 10 U/mL, or first reduced to 25U/mL and then to 10 U/mL thereafter after supplementing the wells withmedium throughout the assay. On day 14 or 17, the cells were harvestedfor flow cytometry analysis.

IgG1-CD27-A-P329R-E345R enhanced expansion of TIL subtypes compared tocontrol cultures treated with IL-2 alone, with the largest relativeincrease in cell count observed for CD8⁺T cells and Tregs, followed byCD4⁺ T cells, and NK cells. For all TIL subsets, expansion was morepronounced with IgG1-CD27-A-P329R-E345R at 1 µg/mL than 10 µg/mL (Table6 and FIG. 23 ).

Table 6. Fold-expansion of IgG1-CD27-A-P329R-E345R-treated TIL

Tumor tissues derived from human NSCLC specimens were cultured withlow-dose IL-2 in the presence or absence of IgG1-CD27-A-P329R-E345R.Absolute cell counts of the indicated cell subsets were determined byflow cytometry after 14 to 17 days of treatment. Fold differences incell numbers for IgG1-CD27-A-P329R-E345R-treated cultures relative tocultures treated with IL-2 are shown. Data shown are from five tumortissues from individual patients tested in five independent experiments.P=0.0236, 1 µg/mL vs. 10 µg/mL IgG1-CD27-A-P329R-E345R (two-way ANOVA).

Cell population All TIL CD4⁺ T cells CD8⁺ T cells Treg NK cellsIgG1-CD27-A-P329R-E345R concentration (µg/mL) 1 10 1 10 1 10 1 10 1 10Patient #578 14.9 2.1 19.3 2.3 19.6 2.9 86.9 2.1 11.3 1.1 Patient #50727.9 5.1 33.7 4.4 107.1 17.4 32.2 11.9 14.4 6.0 Patient #594 0.6 1.5 0.41.0 1.8 2.2 0.4 2.3 0.8 2.6 Patient #592 0.9 0.8 0.4 0.2 2.9 1.7 4.8 2.62.3 1.2 Patient #561 0.8 1.6 0.2 2.9 0.8 1.0 n.d. n.d. 1.1 1.2Average±SD^(a) 11.1± 11.3 2.4± 1.6 13.5± 14.0 2.0± 1.6 32.9± 43.4 6.1±6.6 31.1± 34.5 4.9± 4.1 7.2± 5.8 2.7± 2.0 ^(a)Average and SDcalculations exclude patient #561 for better comparability between cellpopulations. Abbreviations: ANOVA = analysis of variance; n.d. = notdetermined; NK = natural killer; NSCLC = non-small cell lung cancer; SD= standard deviation; TIL = tumor-infiltrating lymphocyte; Treg =regulatory T cell.

Example 26: BRET Analysis to Assess Intermolecular Interactions ofIgG1-CD27-A-P329R-E345R Molecules on the Cell Surface

The capacity of CD27 antibodies harboring the hexamerization-enhancingmutation (E345R) to increase intermolecular Fc-Fc interactions afterbinding to CD27 on the cell surface was determined using bioluminescenceresonance energy transfer (BRET) analysis. This molecularproximity-based assay detects protein interactions by measuring energytransfer from a bioluminescent protein donor to a fluorescent proteinacceptor. Energy transfer occurs only when the donor and acceptor are inclose proximity (<10 nm [Wu and Brand, 1994; Dacres et al, 2012]).

First, cell surface expression of CD27, as well as CD20 and CD37 (aspositive control molecules), was determined on huCD27-K562, a humanchronic myelogenous leukemia cell line genetically modified to stablyexpress human CD27, and on Daudi cells, using an indirectimmunofluorescence assay (QIFIKIT, Agilent Technologies, cat no. K0078).Cells were seeded at 100,000 cells/well and incubated with 10 µg/mLprimary antibody (CD27: IgG1-7730-143-C102S-FEAL; CD20: IgG1-11B8-FEAR;CD37: IgG1-3009-010-FEAR). This was followed by incubation with aFITC-labeled polyclonal goat anti-human IgG (Jackson Immuno Research,cat. no. 109-096-097), in parallel with QIFIKIT beads coated with adefined number of antibody molecules. The number of antibody moleculesper cell was determined by interpolating the measured mean fluorescenceintensity (MFI) of a test sample on the calibration curve generated byplotting the MFI of the individual bead populations against the knownnumber of antibody molecules per bead. Samples were measured on anLSRFortessa Cell Analyzer flow cytometer (BD Biosciences) and analyzedusing FlowJo software.

QiFi analysis showed moderate CD27 expression and high CD20 and CD37expression on Daudi cells, whereas huCD27-K562 cells expressed highlevels of CD27, but no CD20 and CD37 (Table 7).

TABLE 7 Cell surface expression in antibody molecules per cellhuCD27-K562 Daudi CD27 390,373 15,484 CD20 - 180,217 CD37 - 219,663

BRET assay (NanoBRET™ System, Promega, cat no. N1661) was performedessentially according to the manufacturer’s instructions. To generateNanoLuc (donor) and HaloTag (acceptor) tagged antibodies, variable lightchain sequences with either NanoLuc or HaloTag (Table 3, sequences37-44) were prepared by gene synthesis, cloned into appropriateexpression vectors and full-length antibodies produced as described inExample 1. For analysis, 0.5×10⁵ huCD27-K562 or Daudi cells were seededin 96-well round-bottom plates (Greiner Bio-One, cat. no. 650101) in atotal volume of 100 µL. Cells were pelleted by centrifugation (3 min at300xg) and resuspended in 50 µL assay medium (Opti-MEM I [Gibco, cat.no. 11058-021] + 4% FBS [ATCC, cat. no. 30-2020]) containing mixtures ofNanoLuc- or HaloTag-tagged antibody pairs each at a concentration of 5µg/mL. Next, 50 µL HaloTag NanoBret 618 ligand (Promega, cat. no. G980A,1:1000 dilution in assay medium) was added. For each antibody mixture, ano-ligand control sample was prepared in parallel, by adding 50 µLmedium without HaloTag NanoBret 618 ligand. Cells were incubated for 30min at 37° C. in the dark, washed twice with medium and resuspended in100 µL assay medium without FBS. 25 µL NanoBRET NanoGLO substrate(Promega, cat. no. N1571, 1:200 dilution in assay medium without FBS)was added to each well. Plates were shaken for 30 s and 120 µL of eachsample was transferred to an OptiPlate (Perkin Elmer, cat. no. 6005299).An EnVision Multilabel Reader (Perkin Elmer) was used to measure donoremission at 460 nm and acceptor emission at 618 nm.

BRET was calculated in milliBRET units (mBU) = (618 nm_(em)/460 nm_(em))× 1000.

Results are reported as Corrected BRET, which is corrected fordonor-contributed background or bleedthrough, and calculated as: mBUligand - mBU no-ligand control.

The proximity of NanoLuc- and HaloTag-labeled IgG1-CD27-A-P329R-E345Rantibodies after binding CD27 on the cell surface was compared to WTIgG1-CD27-A antibodies carrying the same tags.IgG1-CD20-11B8-E430G-LNLuc and IgG1-CD37-37.3-E430G-LHalo antibodies,containing an E430G mutation that induces hexamerization(WO2019243636A1), were used as a positive control for proximity-inducedBRET. IgG1-CD20-11B8-E430G and IgG1-CD37-37.3-E430G were previouslyshown to form heterohexamers upon binding to cells expressing CD20 andCD37, using molecular proximity assays (Oostindie, S.C. et al,Haematologica, 2019). Nonbinding antibody IgG1-b12-P329R-E345R was usedas a negative control.

As positive and negative controls for BRET signal induction, Daudi cells(high CD20 and CD37 expression) and huCD27-K562 cells (no CD20 and CD37expression) were opsonized with antibody pair IgGl-CD20-11B8-E430G-LNLucand IgG1-CD37-37.3-E430G-LHalo. BRET induction was detected only onDaudi cells, and not on huCD27-K562 cells lacking CD20 and CD37 (FIG. 24). Similarly, a non-binding control antibody pair(IgG1-b12-P329R-E345R-LNLuc + IgG1-b12-P329R-E345R-LHalo) did not induceBRET on either cell line. When huCD27-K562 cells were opsonized with amixture of NanoLuc- and HaloTag-labeled CD27 antibodies bearing thehexamerization-enhancing mutation (IgG1-CD27-A-P329R-E345R-LNLuc +IgG1-CD27-A-P329R-E345R-LHalo), high BRET was detected, while BRET onDaudi cells did not exceed background levels (FIG. 24 ). A mixture ofIgG1-CD27-A-LNLuc and IgG1-CD27-A-LHalo (WT) antibodies inducedconsiderably lower BRET on huCD27-K562 cells compared to CD27 antibodiescarrying the P329R and E345R mutations, and no BRET on Daudi cells.These results indicate that BRET signal was associated with highertarget expression. CD27 expression on huCD27-K562 cells was found to be~26 fold higher than on Daudi cells, while BRET levels for CD27-bindingIgG1-CD27-A-P329R-E345R on huCD27-K562 cells were ~24 fold higher thanon Daudi cells. Mixtures of NanoLuc- and HaloTag-labeled nonbinding andCD27-binding antibody pairs (IgG1-b12-P329R-E345R-LNLuc +IgG1-CD27-A-P329R-E345R-LHalo, and IgG1-CD27-A-P329R-E345R-LNLuc +IgG1-b12-P329R-E345R-LHalo respectively), did not induce BRET on eithercell line. This confirms that observed BRET was dependent onsimultaneous interaction of donor and acceptor antibodies bound to thecell-surface target.

In summary, IgG1-CD27-A-P329R-E345R induced high BRET on huCD27-K562cells compared to its WT variant. This finding confirms enhancedproximity between membrane-bound IgG1-CD27-A-P329R-E345R molecules,compared to its WT variant, consistent with E345R-enhanced Fc-Fcinteractions between cell surface-bound antibodies.

N.B. the experiment described in this example used a variant ofIgG1-CD27-A carrying a F405L mutation, which is functionally irrelevantin the context of this experiment.

Example 27: Binding of IgG1-CD27-A-P329R-E345R to FcγRla⁺ M0 and M1Macrophages

Example 9 assessed binding of IgG1-CD27-A-P329R-E345R to human FcyRvariants using surface plasmon resonance (SPR), showing minimal (FcyRla)or no (FcyRlla, FcyRllb, and FcyRllla) binding to recombinant human IgGFc receptor molecules. This residual FcyRla binding was not sufficientto induce IgG1-CD27-A-P329R-E345R-dependent ADCP of CD27⁺ cells (seeExample 13). To further exclude interactions of IgG1-CD27-A-P329R-E345Rwith FcyRla-positive macrophages, Fc-mediated binding ofIgG1-CD27-A-P329RE345R to M0 and M1 macrophages was determined.

Human CD14⁺ monocytes were isolated from PBMCs from two healthy donorsas described in Example 13, and differentiated into monocyte-derivedmacrophages by culturing the cells in medium (CellGenix, cat. no.20801-0500) supplemented with 50 ng/mL M-CSF (Gibco, cat. no. PHC9501)to obtain M0 macrophages, or 50 ng/mL GM-CSF (Immunotools, cat. no.11343125) for differentiation into M1 macrophages. After 6 days ofculture, M0 and M1 phenotypes were confirmed by FACS analysis accordingto expression of markers as defined in Table 8. Additionally, bothmacrophage subtypes were confirmed to express human Fc receptors FcyRla,FcyRll and FcyRllla (Table 8).

TABLE 8 Phenotype markers M0 M1 CD40 (BD Pharmingen, cat. no. 561211,1:50 dilution) + + CD86 (MACS, cat. no. 30-097-877, 1:50 dilution) + ++CD163 (Biolegend, cat.no. 333612, 1:200 dilution) +/- - CD206(Biolegend, cat. no. 321136, 1:200 dilution) +/- + Fc receptors FcyRla(Biolegend, cat. no. 305006, 1:25 dilution) ++ ++ FcyRll (BD Pharmingen,cat. no. 552883, 1:50 dilution) ++ ++ FcyRllla (BD Pharmingen, cat. no.555407, 1:50 dilution) + +/-

Binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages was comparedto binding of a WT IgG1 antibody (IgG1-b12) with an irrelevantantigen-binding region as a positive control for FcyRla binding, and avariant of the same antibody also carrying the P329R mutation previouslydescribed to reduce interaction with FcyR (IgG1-b12-P329R-E345R). Sincemacrophages should not express CD27, any binding observed ishypothesized to occur via FcyRla, which is the only FcyR that bindsmonovalent IgG. The differentiated macrophages were incubated withIgG1-CD27-A-P329R-E345R or control antibodies (30 µg/mL in DC medium)for 15 min, and PE-labeled polyclonal goat anti-human IgG (JacksonImmuno Research, cat. no. 109-116-097, dilution 1:200, 30 min at 4° C.).After incubation, cells were washed and resuspended in 100 µL FACSbuffer containing nucleus-staining DAPI (BD Pharmingen, cat. no. 564907,1:5000 dilution). Samples were measured on a FACSymphony flow cytometer(BD Biosciences) and analyzed using FlowJo software.

No binding above background (secondary antibody only) to M0 or M1macrophages isolated from two independent donors was observed witheither IgG1-CD27-A-P329R-E345R or control IgG1-b12-P329RE345R (FIG. 25). WT IgG1-b12, which contains an active Fc region, consistently boundto both M0 and M1 macrophages.

In conclusion, the IgG1-CD27-A-P329R-E345R and controlIgG1-b12-P329R-E345R do not bind M0 or M1 macrophages expressing FcyRla,FcyRll and FcyRllla.

1-65. (canceled)
 66. An anti-CD27 antibody, or antigen binding fragmentthereof, that specifically binds to human CD27, wherein the antibody orantigen binding fragment further comprises a human heavy chain constantregion IgG, wherein the amino acid residue at the position correspondingto position E345 or E430 in the human IgG1 heavy chain according to Eunumbering is selected from the group comprising: A, C, D, F, G, H, I, K,L, M, N, Q, R, S, T, V, W and Y..
 67. The antibody of claim 66, whereinthe amino acid residue at the position corresponding to position E345 inthe human IgG1 heavy chain according to Eu numbering is R.
 68. Theantibody of claim 66, wherein the amino acid residue at the positioncorresponding to position E430 in the human IgG1 heavy chain accordingto Eu numbering is G.
 69. The antibody of claim 66, wherein the aminoacid residue at the position corresponding to position P329 in the humanIgG1 heavy chain according to Eu numbering is R.
 70. The antibody ofclaim 66, wherein the amino acid residue at the positions correspondingto position E345 and P329 in a human IgG1 heavy chain according to Eunumbering are both R.
 71. A pharmaceutical composition comprising theanti-CD27 antibody of claim 66 and a pharmaceutically acceptableexcipient, diluent, or carrier.
 72. An anti-CD27 antibody, or antigenbinding fragment thereof, that specifically binds to human CD27, whereinthe antibody or antigen binding fragment comprises a heavy chainvariable (VH) region CDR1, CDR2, and CDR3 comprising the sequences asset forth in SEQ ID NOs: 5, 6, and 7, respectively, and a light chainvariable (VL) region CDR1, CDR2, and CDR3 comprising the sequences asset forth in SEQ ID NO: 9, 10 and 11, respectively.
 73. The antibody ofclaim 72, wherein the antibody comprises the heavy chain constant regioncomprising a sequence selected from the group comprising: SEQ ID Nos 12,13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and36.
 74. The antibody of claim 72, wherein the antibody comprises theheavy chain constant region comprising the sequence as set forth in SEQID No
 15. 75. A pharmaceutical composition comprising the anti-CD27antibody of claim 72 and a pharmaceutically acceptable carrier.
 76. Ananti-CD27antibody, or antigen binding fragment thereof, thatspecifically binds to human CD27, the antibody or antigen bindingfragment comprising VH and VL regions comprising the sequences as setforth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.
 77. The antibodyof claim 75, wherein the anti-CD27 antibody further comprises a humanheavy chain constant region IgG
 1. 78. The antibody of claim 75, whereinthe antibody comprises the heavy chain constant region comprising asequence selected from the group comprising: SEQ ID Nos 12, 13, 14, 15,18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and
 36. 79. Theantibody of claim 75, wherein the antibody further comprises: a. Theheavy chain constant region comprising the amino acid sequence set forthin SEQ ID No: 15; and b. The light chain constant region comprising theamino acid sequence set forth in SEQ ID No:
 17. 80. A pharmaceuticalcomposition comprising the anti-CD27 antibody of claim 75 and apharmaceutically acceptable excipient, diluent, or carrier.
 81. Ananti-CD27 antibody that specifically binds to human CD27, wherein theantibody comprises a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 35 and a light chain comprising the amino acidsequence set forth in SEQ ID NO:
 25. 82. A pharmaceutical compositioncomprising the anti-CD27 antibody of claim 81 and a pharmaceuticallyacceptable excipient, diluent, or carrier.
 83. A method of treatingcancer, an inflammatory and/or autoimmune disease or disorder, themethod comprising administering to the subject the pharmaceuticalcomposition of claim
 71. 84. A method of treating cancer, aninflammatory and/or autoimmune disease or disorder, the methodcomprising administering to the subject the pharmaceutical compositionof claim
 75. 85. A method of treating cancer, an inflammatory and/orautoimmune disease or disorder, the method comprising administering tothe subject the pharmaceutical composition of claim
 80. 86. A method oftreating cancer, an inflammatory and/or autoimmune disease or disorder,the method comprising administering to the subject the pharmaceuticalcomposition of claim
 82. 87. A polynucleotide sequence, or set ofpolynucleotides, encoding the anti-CD27 antibody of claim 66, or a hostcell comprising said polynucleotide sequence of set of polynucleotides.88. A polynucleotide sequence, or set of polynucleotides, encoding theanti-CD27 antibody of claim 72, or a host cell comprising saidpolynucleotide sequence of set of polynucleotides.
 89. A polynucleotidesequence, or set of polynucleotides, encoding the anti-CD27 antibody ofclaim 76, or a host cell comprising said polynucleotide sequence of setof polynucleotides.
 90. A polynucleotide sequence, or set ofpolynucleotides, encoding the anti-CD27 antibody of claim 81, or a hostcell comprising said polynucleotide sequence of set of polynucleotides.91. A method of making an anti-CD27 antibody comprising culturing thehost cell of claim 87 under conditions to produce the antibody andrecovering the antibody.
 92. A method of making an anti-CD27 antibodycomprising culturing the host cell of claim 88 under conditions toproduce the antibody and recovering the antibody.
 93. A method of makingan anti-CD27 antibody comprising culturing the host cell of claim 89under conditions to produce the antibody and recovering the antibody.94. A method of making an anti-CD27 antibody comprising culturing thehost cell of claim 89 under conditions to produce the antibody andrecovering the antibody.
 95. An anti-idiotypic antibody, which binds tothe antibody or antigen binding fragment of claim 66.